Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof

ABSTRACT

An antisense molecule capable of binding to a selected target site to induce exon skipping in the dystrophin gene, as set forth in SEQ ID NO: 1 to 202.

FIELD OF THE INVENTION

The present invention relates to novel antisense compounds andcompositions suitable for facilitating exon skipping. It also providesmethods for inducing exon skipping using the novel antisense compoundsas well as therapeutic compositions adapted for use in the methods ofthe invention.

BACKGROUND ART

Significant effort is currently being expended researching methods forsuppressing or compensating for disease-causing mutations in genes.Antisense technologies are being developed using a range of chemistriesto affect gene expression at a variety of different levels(transcription, splicing, stability, translation). Much of that researchhas focused on the use of antisense compounds to correct or compensatefor abnormal or disease-associated genes in a myriad of differentconditions.

Antisense molecules are able to inhibit gene expression with exquisitespecificity and because of this many research efforts concerningoligonucleotides as modulators of gene expression have focused oninhibiting the expression of targeted genes such as oncogenes or viralgenes. The antisense oligonucleotides are directed either against RNA(sense strand) or against DNA where they form triplex structuresinhibiting transcription by RNA polymerase II. To achieve a desiredeffect in specific gene down-regulation, the oligonucleotides musteither promote the decay of the targeted mRNA or block translation ofthat mRNA, thereby effectively preventing de novo synthesis of theundesirable target protein.

Such techniques are not useful where the object is to up-regulateproduction of the native protein or compensate for mutations whichinduce premature termination of translation such as nonsense orframe-shifting mutations. Furthermore, in cases where a normallyfunctional protein is prematurely terminated because of mutationstherein, a means for restoring some functional protein productionthrough antisense technology has been shown to be possible throughintervention during the splicing processes (Sierakowska H, et al.,(1996) Proc Natl Acad Sci USA 93,12840-12844; Wilton S D, et al., (1999)Neuromusc Disorders 9, 330-338; van Deutekom J C et al., (2001) HumanMol Genet 10, 1547-1554). In these cases, the defective gene transcriptshould not be subjected to targeted degradation so the antisenseoligonucleotide chemistry should not promote target mRNA decay.

In a variety of genetic diseases, the effects of mutations on theeventual expression of a gene can be modulated through a process oftargeted exon skipping during the splicing process. The splicing processis directed by complex multi-particle machinery that brings adjacentexon-intron junctions in pre-mRNA into close proximity and performscleavage of phosphodiester bonds at the ends of the introns with theirsubsequent reformation between exons that are to be spliced together.This complex and highly precise process is mediated by sequence motifsin the pre-mRNA that are relatively short semi-conserved RNA segments towhich bind the various nuclear splicing factors that are then involvedin the splicing reactions. By changing the way the splicing machineryreads or recognises the motifs involved in pre-mRNA processing, it ispossible to create differentially spliced mRNA molecules. It has nowbeen recognised that the majority of human genes are alternativelyspliced during normal gene expression, although the mechanisms invokedhave not been identified. Using antisense oligonucleotides, it has beenshown that errors and deficiencies in a coded mRNA could be bypassed orremoved from the mature gene transcripts.

In nature, the extent of genetic deletion or exon skipping in thesplicing process is not fully understood, although many instances havebeen documented to occur, generally at very low levels (Sherrat T G, etal., (1993) Am J Hum Genet 53, 1007-1015). However, it is recognisedthat if exons associated with disease-causing mutations can bespecifically deleted from some genes, a shortened protein product cansometimes be produced that has similar biological properties of thenative protein or has sufficient biological activity to ameliorate thedisease caused by mutations associated with the target exon (Lu Q L, etal., (2003) Nature Medicine 9, 1009-1014; Aartsma-Rus A et al., (2004)Am J Hum Genet 74: 83-92).

This process of targeted exon skipping is likely to be particularlyuseful in long genes where there are many exons and introns, where thereis redundancy in the genetic constitution of the exons or where aprotein is able to function without one or more particular exons (e.g.with the dystrophin gene, which consists of 79 exons; or possibly somecollagen genes which encode for repeated blocks of sequence or the hugenebulin or titin genes which are comprised of ˜80 and over 370 exons,respectively).

Efforts to redirect gene processing for the treatment of geneticdiseases associated with truncations caused by mutations in variousgenes have focused on the use of antisense oligonucleotides that either:(1) fully or partially overlap with the elements involved in thesplicing process; or (2) bind to the pre-mRNA at a position sufficientlyclose to the element to disrupt the binding and function of the splicingfactors that would normally mediate a particular splicing reaction whichoccurs at that element (e.g., binds to the pre-mRNA at a position within3, 6, or 9 nucleotides of the element to be blocked).

For example, modulation of mutant dystrophin pre-mRNA splicing withantisense oligoribonucleotides has been reported both in vitro and invivo. In one type of dystrophin mutation reported in Japan, a 52-basepair deletion mutation causes exon 19 to be removed with the flankingintrons during the splicing process (Matsuo et al., (1991) J ClinInvest. 87:2127-2131). An in vitro minigene splicing system has beenused to show that a 31-mer 2′-O-methyl oligoribonucleotide complementaryto the 5′ half of the deleted sequence in dystrophin Kobe exon 19inhibited splicing of wild-type pre-mRNA (Takeshima et al. (1995), J.Clin. Invest., 95, 515-520). The same oligonucleotide was used to induceexon skipping from the native dystrophin gene transcript in humancultured lymphoblastoid cells.

Dunckley et al., (1997) Nucleosides & Nucleotides, 16, 1665-1668described in vitro constructs for analysis of splicing around exon 23 ofmutated dystrophin in the mdx mouse mutant, a model for musculardystrophy. Plans to analyse these constructs in vitro using 2′ modifiedoligonucleotides targeted to splice sites within and adjacent to mousedystrophin exon 23 were discussed, though no target sites or sequenceswere given.

2′-O-methyl oligoribonucleotides were subsequently reported to correctdystrophin deficiency in myoblasts from the mdx mouse from this group.An antisense oligonucleotide targeted to the 3′ splice site of murinedystrophin intron 22 was reported to cause skipping of the mutant exonas well as several flanking exons and created a novel in-framedystrophin transcript with a novel internal deletion. This mutateddystrophin was expressed in 1-2% of antisense treated mdx myotubes. Useof other oligonucleotide modifications such as 2′-O-methoxyethylphosphodiesters are described (Dunckley et al. (1998) Human Mol.Genetics, 5, 1083-90).

Thus, antisense molecules may provide a tool in the treatment of geneticdisorders such as Duchenne Muscular Dystrophy (DMD). However, attemptsto induce exon skipping using antisense molecules have had mixedsuccess. Studies on dystrophin exon 19, where successful skipping ofthat exon from the dystrophin pre-mRNA was achieved using a variety ofantisense molecules directed at the flanking splice sites or motifswithin the exon involved in exon definition as described by Errington etal. (2003) J Gen Med 5, 518-527”.

In contrast to the apparent ease of exon 19 skipping, the first reportof exon 23 skipping in the mdx mouse by Dunckley et al., (1998) is nowconsidered to be reporting only a naturally occurring revertanttranscript or artefact rather than any true antisense activity. Inaddition to not consistently generating transcripts missing exon 23,Dunckley et al., (1998) did not show any time course of induced exonskipping, or even titration of antisense oligonucleotides, todemonstrate dose dependent effects where the levels of exon skippingcorresponded with increasing or decreasing amounts of antisenseoligonucleotide. Furthermore, this work could not be replicated by otherresearchers.

The first example of specific and reproducible exon skipping in the mdxmouse model was reported by Wilton et al., (1999) NeuromuscularDisorders 9, 330-338. By directing an antisense molecule to the donorsplice site, consistent and efficient exon 23 skipping was induced inthe dystrophin mRNA within 6 hours of treatment of the cultured cells.Wilton et al., (1999), also describe targeting the acceptor region ofthe mouse dystrophin pre-mRNA with longer antisense oligonucleotides andbeing unable to repeat the published results of Dunckley et al., (1998).No exon skipping, either 23 alone or multiple removal of severalflanking exons, could be reproducibly detected using a selection ofantisense oligonucleotides directed at the acceptor splice site ofintron 22.

While the first antisense oligonucleotide directed at the intron 23donor splice site induced consistent exon skipping in primary culturedmyoblasts, this compound was found to be much less efficient inimmortalized cell cultures expressing higher levels of dystrophin.However, with refined targeting and antisense oligonucleotide design,the efficiency of specific exon removal was increased by almost an orderof magnitude (see Mann C J et al., (2002) J Gen Med 4, 644-654).

Thus, there remains a need to provide antisense oligonucleotides capableof binding to and modifying the splicing of a target nucleotidesequence. Simply directing the antisense oligonucleotides to motifspresumed to be crucial for splicing is no guarantee of the efficacy ofthat compound in a therapeutic setting.

SUMMARY OF THE INVENTION

The present invention provides antisense molecule compounds andcompositions suitable for binding to RNA motifs involved in the splicingof pre-mRNA that are able to induce specific and efficient exon skippingand a method for their use thereof.

The choice of target selection plays a crucial role in the efficiency ofexon skipping and hence its subsequent application of a potentialtherapy. Simply designing antisense molecules to target regions ofpre-mRNA presumed to be involved in splicing is no guarantee of inducingefficient and specific exon skipping. The most obvious or readilydefined targets for splicing intervention are the donor and acceptorsplice sites although there are less defined or conserved motifsincluding exonic splicing enhancers, silencing elements and branchpoints.

The acceptor and donor splice sites have consensus sequences of about 16and 8 bases respectively (see FIG. 1 for schematic representation ofmotifs and domains involved in exon recognition, intron removal and thesplicing process).

According to a first aspect, the invention provides antisense moleculescapable of binding to a selected target to induce exon skipping.

For example, to induce exon skipping in exons 3 to 8, 10 to 16, 19 to40, 42 to 44, 46, 47, and 50 to 53 in the Dystrophin gene transcript theantisense molecules are preferably selected from the group listed inTable 1A.

In a further example, it is possible to combine two or more antisenseoligonucleotides of the present invention together to induce multipleexon skipping in exons 19-20, and 53. This is a similar concept totargeting of a single exon. A combination or “cocktail” of antisenseoligonucleotides are directed at adjacent exons to induce efficient exonskipping.

In another example, to induce exon skipping in exons 19-20, 31, 34 and53 it is possible to improve exon skipping of a single exon by joiningtogether two or more antisense oligonucleotide molecules. This conceptis termed by the inventor as a “weasel”, an example of a cunninglydesigned antisense oligonucleotide. A similar concept has been describedin Aartsma-Rus A et al., (2004) Am J Hum Genet 74: 83-92).

According to a second aspect, the present invention provides antisensemolecules selected and or adapted to aid in the prophylactic ortherapeutic treatment of a genetic disorder comprising at least anantisense molecule in a form suitable for delivery to a patient.

According to a third aspect, the invention provides a method fortreating a patient suffering from a genetic disease wherein there is amutation in a gene encoding a particular protein and the affect of themutation can be abrogated by exon skipping, comprising the steps of: (a)selecting an antisense molecule in accordance with the methods describedherein; and (b) administering the molecule to a patient in need of suchtreatment.

The invention also addresses the use of purified and isolated antisenseoligonucleotides of the invention, for the manufacture of a medicamentfor treatment of a genetic disease.

The invention further provides a method of treating a conditioncharacterised by Duchenne muscular dystrophy, which method comprisesadministering to a patient in need of treatment an effective amount ofan appropriately designed antisense oligonucleotide of the invention,relevant to the particular genetic lesion in that patient. Further, theinvention provides a method for prophylactically treating a patient toprevent or at least minimise Duchene muscular dystrophy, comprising thestep of: administering to the patient an effective amount of anantisense oligonucleotide or a pharmaceutical composition comprising oneor more of these biological molecules.

The invention also provides kits for treating a genetic disease, whichkits comprise at least a antisense oligonucleotide of the presentinvention, packaged in a suitable container and instructions for itsuse.

Other aspects and advantages of the invention will become apparent tothose skilled in the art from a review of the ensuing description, whichproceeds with reference to the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of motifs and domains involved in exonrecognition, intron removal and the splicing process.

FIG. 2. Diagrammatic representation of the concept of antisenseoligonucleotide induced exon skipping to by-pass disease-causingmutations (not drawn to scale). The hatched box represents an exoncarrying a mutation that prevents the translation of the rest of themRNA into a protein. The solid black bar represents an antisenseoligonucleotide that prevents inclusion of that exon in the mature mRNA.

FIG. 3 Gel electrophoresis showing differing efficiencies of twoantisense molecules directed at exon 8 acceptor splice site. Thepreferred compound [H8A(−06+18)] induces strong and consistent exonskipping at a transfection concentration of 20 nanomolar in culturednormal human muscle cells. The less preferred antisense oligonucleotide[H8A(−06+14)] also induces efficient exon skipping, but at much higherconcentrations. Other antisense oligonucleotides directed at exon 8either only induced lower levels of exon skipping or no detectableskipping at all (not shown).

FIG. 4 Gel electrophoresis showing differing efficiencies of twoantisense molecules directed at internal domains within exon 7,presumably exon splicing enhancers. The preferred compound [H7A(+45+67)]induces strong and consistent exon skipping at a transfectionconcentration of 20 nanomolar in cultured human muscle cells. The lesspreferred antisense oligonucleotide [H7A(+2+26)] induces only low levelsof exon skipping at the higher transfection concentrations. Otherantisense oligonucleotides directed at exon 7 either only induced lowerlevels of exon skipping or no detectable skipping at all (not shown).

FIG. 5 Gel electrophoresis showing an example of low efficiency exon 6skipping using two non-preferred antisense molecules directed at humanexon 6 donor splice site. Levels of induced exon 6 skipping are eithervery low [H6D(+04-21)] or almost undetectable [H6D(+18-04)]. These areexamples of non-preferred antisense oligonucleotides to demonstrate thatantisense oligonucleotide design plays a crucial role in the efficacy ofthese compounds.

FIG. 6 Gel electrophoresis showing strong and efficient human exon 6skipping using an antisense molecules [H6A(+69+91)] directed at an exon6 internal domain, presumably an exon splicing enhancer. This preferredcompound induces consistent exon skipping at a transfectionconcentration of 20 nanomolar in cultured human muscle cells.

FIG. 7 Gel electrophoresis showing strong human exon 4 skipping using anantisense molecule H4A(+13+32) directed at an exon 6 internal domain,presumably an exon splicing enhancer. This preferred compound inducesstrong and consistent exon skipping at a transfection concentration of20 nanomolar in cultured human muscle cells.

FIG. 8 Gel electrophoresis showing (8B) strong human exon 11 skippingusing antisense molecule H11A(+75+97) directed at an exon 11 internaldomain; and (8B) strong human exon 12 skipping using antisense moleculeH12A(+52+75) directed at exon 12 internal domain.

FIG. 9 Gel electrophoresis showing (9A) strong human exon 15 skippingusing antisense molecules H15A(+48+71) and H15A(−12+19) directed at anexon 15 internal domain; and (9B) strong human exon 16 skipping usingantisense molecules H16A(−12+19) and H16A(−06+25).

FIG. 10 Gel electrophoresis showing human exon 19/20 skipping usingantisense molecules H20A(+44+71) and H20A(+149+170) directed at an exon20 and a “cocktail” of antisense oligonucleotides H19A(+35+65,H20A(+44+71) and H20A(+149+170) directed at exons 19/20.

FIG. 11 Gel electrophoresis showing human exon 19/20 skipping using“weasels” directed at exons 19 and 20.

FIG. 12 Gel electrophoresis showing exon 22 skipping using antisensemolecules H22A(+125+106), H22A(+47+69), H22A(+80+101) and H22D(+13−11)directed at exon 22.

FIG. 13 Gel electrophoresis showing exon 31 skipping using antisensemolecules H31D(+01−25) and H31D(+03−22); and a “cocktail” of antisensemolecules directed at exon 31.

FIG. 14 Gel electrophoresis showing exon 33 skipping using antisensemolecules H33A(+30+56) and H33A(+64+88) directed at exon 33.

FIG. 15 Gel electrophoresis showing exon 35 skipping using antisensemolecules H35A(+141+161), H35A(+116+135), and H35A(+24+43) and a“cocktail of two antisense molecules, directed at exon 35.

FIG. 16 Gel electrophoresis showing exon 36 skipping using antisensemolecules H32A(+49+73) and H36A(+26+50) directed at exon 36.

FIG. 17 Gel electrophoresis showing exon 37 skipping using antisensemolecules H37A(+82+105) and H37A(+134+157) directed at exon 37.

FIG. 18 Gel electrophoresis showing exon 38 skipping using antisensemolecule H38A(+88+112) directed at exon 38.

FIG. 19 Gel electrophoresis showing exon 40 skipping using antisensemolecule H40A(−05+17) directed at exon 40.

FIG. 20 Gel electrophoresis showing exon 42 skipping using antisensemolecule H42A(−04+23) directed at exon 42.

FIG. 21 Gel electrophoresis showing exon 46 skipping using antisensemolecule H46A(+86+115) directed at exon 46

FIG. 22 Gel electrophoresis showing exon 51, exon 52 and exon 53skipping using various antisense molecules directed at exons 51, 52 and53, respectively. A “cocktail” of antisense molecules is also showndirected at exon 53.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

TABLE 1ADescription of 2′-O-methyl phosphorothioate antisense oligonucleotidesthat have been used to date to study induced exon skipping during theprocessing of the dystrophin pre-mRNA. Since these 2′-O-methylantisense oligonucleotides are more RNA-like, U represents uracil. With other antisense chemistries such as pepide nucleic acids ormorpholinos, these U bases may be shown as “T”. SEQ ID SEQUENCENUCLEOTIDE SEQUENCE (5′ - 3′) 1 H8A(−06 +18)GAU AGG UGG UAU CAA CAU CUG UAA 2 H8A(−03 +18)GAU AGG UGG UAU CAA CAU CUG 3 H8A(−07 +18)GAU AGG UGG UAU CAA CAU CUG UAA G 4 H8A(−06 +14)GGU GGU AUC AAC AUC UGU AA 5 H8A(−10 +10) GUA UCA ACA UCU GUA AGC AC 6H7A(+45 +67) UGC AUG UUC CAG UCG UUG UGU GG 7 H7A(+02 +26)CAC UAU UCC AGU CAA AUA GGU CUG G 8 H7D(+15 −10)AUU UAC CAA CCU UCA GGA UCG AGU A 9 H7A(−18 +03)GGC CUA AAA CAC AUA CAC AUA 10 C6A(−10 +10) CAU UUU UGA CCU ACA UGU GG11 C6A(−14 +06) UUU GAC CUA CAU GUG GAA AG 12 C6A(−14 +12)UAC AUU UUU GAC CUA CAU GUG GAA AG 13 C6A(−13 +09)AUU UUU GAC CUA CAU GGG AAA G 14 CH6A(+69 +91)UAC GAG UUG AUU GUC GGA CCC AG 15 C6D(+12 −13)GUG GUC UCC UUA CCU AUG ACU GUG G 16 C6D(+06 −11) GGU CUC CUU ACC UAU GA17 H6D(+04 −21) UGU CUC AGU AAU CUU CUU ACC UAU 18 H6D(+18 −04)UCU UAC CUA UGA CUA UGG AUG AGA 19 H4A(+13 +32)GCA UGA ACU CUU GUG GAU CC 20 H4D(+04 −16) CCA GGG UAC UAC UUA CAU UA 21H4D(−24 −44) AUC GUG UGU CAC AGC AUC CAG 22 H4A(+11 +40)UGU UCA GGG CAU GAA CUC UUG UGG AUC CUU 23 H3A(+30 +60)UAG GAG GCG CCU CCC AUC CUG UAG GUC ACU G 24 H3A(+35 +65)AGG UCU AGG AGG CGC CUC CCA UCC UGU AGG U 25 H3A(+30 +54)GCG CCU CCC AUC CUG UAG GUC ACU G 26 H3D(+46 −21)CUU CGA GGA GGU CUA GGA GGC GCC UC 27 H3A(+30 +50)CUC CCA UCC UGU AGG UCA CUG 28 H3D(+19 −03)UAC CAG UUU UUG CCC UGU CAG G 29 H3A(−06 +20)UCA AUA UGC UGC UUC CCA AAC UGA AA 30 H3A(+37 +61)CUA GGA GGC GCC UCC CAU CCU GUA G 31 H5A(+20 +50)UUA UGA UUU CCA UCU ACG AUG UCA GUA CUU C 32 H5D(+25 −05)CUU ACC UGC CAG UGG AGG AUU AUA UUC CAA A 33 H5D(+10 −15)CAU CAG GAU UCU UAC CUG CCA GUG G 34 H5A(+10 +34)CGA UGU CAG UAC UUC CAA UAU UCA C 35 H5D(−04 −21)ACC AUU CAU CAG GAU UCU 36 H5D(+16 −02) ACC UGC CAG UGG AGG AUU 37H5A(−07 +20) CCA AUA UUC ACU AAA UCA ACC UGU UAA 38 H5D(+18 −12)CAG GAU UCU UAC CUG CCA GUG GAG GAU UAU 39 H5A(+05 +35)ACG AUG UCA GUA CUU CCA AUA UUC ACU AAA U 40 H5A(+15 +45)AUU UCC AUC UAC GAU GUC AGU ACU UCC AAU A 41 H10A(−05 +16)CAG GAG CUU CCA AAU GCU GCA 42 H10A(−05 +24)CUU GUC UUC AGG AGC UUC CAA AUG CUG CA 43 H10A(+98 +119)UCC UCA GCA GAA AGA AGC CAC G 44 H10A(+130 +149)UUA GAA AUC UCU CCU UGU GC 45 H10A(−33 −14) UAA AUU GGG UGU UAC ACA AU46 H11D(+26 +49) CCC UGA GGC AUU CCC AUC UUG AAU 47 H11D(+11 −09)AGG ACU UAC UUG CUU UGU UU 48 H11A(+118 +140)CUU GAA UUU AGG AGA UUC AUC UG 49 H11A(+75 +97)CAU CUU CUG AUA AUU UUC CUG UU 50 H12A(+52 +75)UCU UCU GUU UUU GUU AGC CAG UCA 51 H12A(−10 +10)UCU AUG UAA ACU GAA AAU UU 52 H12A(+11 +30) UUC UGG AGA UCC AUU AAA AC53 H13A(+77 +100) CAG CAG UUG CGU GAU CUC CAC UAG 54 H13A(+55 +75)UUC AUC AAC UAC CAC CAC CAU 55 H13D(+06 −19)CUA AGC AAA AUA AUC UGA CCU UAA G 56 H14A(+37 +64)CUU GUA AAA GAA CCC AGC GGU CUU CUG U 57 H14A(+14 +35)CAU CUA CAG AUG UUU GCC CAU C 58 H14A(+51 +73)GAA GGA UGU CUU GUA AAA GAA CC 59 H14D(−02 +18)ACC UGU UCU UCA GUA AGA CG 60 H14D(+14 −10)CAU GAC ACA CCU GUU CUU CAG UAA 61 H14A(+61 +80)CAU UUG AGA AGG AUG UCU UG 62 H14A(−12 +12)AUC UCC CAA UAC CUG GAG AAG AGA 63 H15A(−12 +19)GCC AUG CAC UAA AAA GGC ACU GCA AGA CAU U 64 H15A(+48 +71)UCU UUA AAG CCA GUU GUG UGA AUC 65 H15A(+08 +28)UUU CUG AAA GCC AUG CAC UAA 66 H15D(+17 −08)GUA CAU ACG GCC AGU UUU UGA AGA C 67 H16A(−12 +19)CUA GAU CCG CUU UUA AAA CCU GUU AAA ACA A 68 H16A(−06 +25)UCU UUU CUA GAU CCG CUU UUA AAA CCU GUU A 69 H16A(−06 +19)CUA GAU CCG CUU UUA AAA CCU GUU A 70 H16A(+87 +109)CCG UCU UCU GGG UCA CUG ACU UA 71 H16A(−07 +19)CUA GAU CCG CUU UUA AAA CCU GUU AA 72 H16A(−07 +13)CCG CUU UUA AAA CCU GUU AA 73 H16A(+12 +37)UGG AUU GCU UUU UCU UUU CUA GAU CC 74 H16A(+92 +116)CAU GCU UCC GUC UUC UGG GUC ACU G 75 H16A(+45 +67)G AUC UUG UUU GAG UGA AUA CAG U 76 H16A(+105 +126)GUU AUC CAG CCA UGC UUC CGU C 77 H16D(+05 −20)UGA UAA UUG GUA UCA CUA ACC UGU G 78 H16D(+12 −11)GUA UCA CUA ACC UGU GCU GUA C 79 H19A(+35 +53)CAG CAG UAG UUG UCA UCU GC 80 H19A(+35 +65)GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U 81 H20A(+44 +71)CUG GCA GAA UUC GAU CCA CCG GCU GUU C 82 H20A(+149 +170)CAG CAG UAG UUG UCA UCU GCU C 83 H20A(+185 +203)UGA UGG GGU GGU GGG UUG G 84 H20A(−08 +17)AUC UGC AUU AAC ACC CUC UAG AAA G 85 H20A(+30 +53)CCG GCU GUU CAG UUG UUC UGA GGC 86 H20A(−11 +17)AUC UGC AUU AAC ACC CUC UAG AAA GAA A 87 H20D(+08 −20)GAA GGA GAA GAG AUU CUU ACC UUA CAA A 88 H20A(+44 +63)AUU CGA UCC ACC GGC UGU UC 89 H20A(+149 +168 CUG CUG GCA UCU UGC AGU U90 H21A(−06 +16) GCC GGU UGA CUU CAU CCU GUG C 91 H21A(+85 +106)CUG CAU CCA GGA ACA UGG GUC C 92 H21A(+85 +108)GUC UGC AUC CAG GAA CAU GGG UC 93 H21A(+08 +31)GUU GAA GAU CUG AUA GCC GGU UGA 94 H21D(+18 −07)UAC UUA CUG UCU GUA GCU CUU UCU 95 H22A(+22 +45)CAC UCA UGG UCU CCU GAU AGC GCA 96 H22A(+125 +106)CUG CAA UUC CCC GAG UCU CUG C 97 H22A(+47 +69)ACU GCU GGA CCC AUG UCC UGA UG 98 H22A(+80 +101)CUA AGU UGA GGU AUG GAG AGU 99 H22D(+13 −11)UAU UCA CAG ACC UGC AAU UCC CC 100 H23A(+34 +59)ACA GUG GUG CUG AGA UAG UAU AGG CC 101 H23A(+18 +39)UAG GCC ACU UUG UUG CUC UUG C 102 H23A(+72 +90)UUC AGA GGG CGC UUU CUU C 103 H24A(+48 +70)GGG CAG GCC AUU CCU CCU UCA GA 104 H24A(−02 +22)UCU UCA GGG UUU GUA UGU GAU UCU 105 H25A(+9 +36)CTG GGC UGA AUU GUC UGA AUA UCA CUG 106 H25A(+131 +156)CUG UUG GCA CAU GUG AUC CCA CUG AG 107 H25D(+16 −08)GUC UAU ACC UGU UGG CAC AUG UGA 108 H26A(+132 +156)UGC UUU CUG UAA UUC AUC UGG AGU U 109 H26A(−07 +19)CCU CCU UUC UGG CAU AGA CCU UCC AC 110 H26A(+68 +92)UGU GUC AUC CAU UCG UGC AUC UCU G 111 H27A(+82 +106)UUA AGG CCU CUU GUG CUA CAG GUG G 112 H27A(−4 +19)GGG CCU CUU CUU UAG CUC UCU GA 113 H27D(+19 −03)GAC UUC CAA AGU CUU GCA UUU C 114 H28A(−05 +19)GCC AAC AUG CCC AAA CUU CCU AAG 115 H28A(+99 +124)CAG AGA UUU CCU CAG CUC CGC CAG GA 116 H28D(+16 −05)CUU ACA UCU AGC ACC UCA GAG 117 H29A(+57 +81)UCC GCC AUC UGU UAG GGU CUG UGC C 118 H29A(+18 +42)AUU UGG GUU AUC CUC UGA AUG UCG C 119 H29D(+17 −05)CAU ACC UCU UCA UGU AGU UCU C 120 H30A(+122 +147)CAU UUG AGC UGC GUC CAC CUU GUC UG 121 H30A(+25 +50)UCC UGG GCA GAC UGG AUG CUC UGU UC 122 H30D(+19 −04)UUG CCU GGG CUU CCU GAG GCA UU 123 H31D(+06 −18)UUC UGA AAU AAC AUA UAC CUG UGC 124 H31D(+03 −22)UAG UUU CUG AAA UAA CAU AUA CCU G 125 H31A(+05 +25)GAC UUG UCA AAU CAG AUU GGA 126 H31D(+04 −20)GUU UCU GAA AUA ACA UAU ACC UGU 127 H32D(+04 −16)CAC CAG AAA UAC AUA CCA CA 128 H32A(+151 +170)CAA UGA UUU AGC UGU GAC UG 129 H32A(+10 +32)CGA AAC UUC AUG GAG ACA UCU UG 130 H32A(+49 +73)CUU GUA GAC GCU GCU CAA AAU UGG C 131 H33D(+09 −11)CAU GCA CAC ACC UUU GCU CC 132 H33A(+53 +76)UCU GUA CAA UCU GAC GUC CAG UCU 133 H33A(+30 +56)GUC UUU AUC ACC AUU UCC ACU UCA GAC 134 H33A(+64 +88)CCG UCU GCU UUU UCU GUA CAA UCU G 135 H34A(+83 +104)UCC AUA UCU GUA GCU GCC AGC C 136 H34A(+143 +165)CCA GGC AAC UUC AGA AUC CAA AU 137 H34A(−20 +10)UUU CUG UUA CCU GAA AAG AAU UAU AAU GAA 138 H34A(+46 +70)CAU UCA UUU CCU UUC GCA UCU UAC G 139 H34A(+95 +120)UGA UCU CUU UGU CAA UUC CAU AUC UG 140 H34D(+10 −20)UUC AGU GAU AUA GGU UUU ACC UUU CCC CAG 141 H34A(+72 +96)CUG UAG CUG CCA GCC AUU CUG UCA AG 142 H35A(+141 +161)UCU UCU GCU CGG GAG GUG ACA 143 H35A(+116 +135)CCA GUU ACU AUU CAG AAG AC 144 H35A(+24 +43) UCU UCA GGU GCA CCU UCU GU145 H36A(+26 +50) UGU GAU GUG GUC CAC AUU CUG GUC A 146 H36A(−02 +18)CCA UGU GUU UCU GGU AUU CC 147 H37A(+26 +50)CGU GUA GAG UCC ACC UUU GGG CGU A 148 H37A(+82 +105)UAC UAA UUU CCU GCA GUG GUC ACC 149 H37A(+134 +157)UUC UGU GUG AAA UGG CUG CAA AUC 150 H38A(−01 +19)CCU UCA AAG GAA UGG AGG CC 151 H38A(+59 +83)UGC UGA AUU UCA GCC UCC AGU GGU U 152 H38A(+88 +112)UGA AGU CUU CCU CUU UCA GAU UCA C 153 H39A(+62 +85)CUG GCU UUC UCU CAU CUG UGA UUC 154 H39A(+39 +58)GUU GUA AGU UGU CUC CUC UU 155 H39A(+102 +121)UUG UCU GUA ACA GCU GCU GU 156 H39D(+10 −10) GCU CUA AUA CCU UGA GAG CA157 H40A(−05 +17) CUU UGA GAC CUC AAA UCC UGU U 158 H40A(+129 +153)CUU UAU UUU CCU UUC AUC UCU GGG C 159 H42A(−04 +23)AUC GUU UCU UCA CGG ACA GUG UGC UGG 160 H42A(+86 +109)GGG CUU GUG AGA CAU GAG UGA UUU 161 H42D(+19 −02)A CCU UCA GAG GAC UCC UCU UGC 162 H43D(+10 −15)UAU GUG UUA CCU ACC CUU GUC GGU C 163 H43A(+101 +120)GGA GAG AGC UUC CUG UAG CU 164 H43A(+78 +100)UCA CCC UUU CCA CAG GCG UUG CA 165 H44A(+85 +104)UUU GUG UCU UUC UGA GAA AC 166 H44D(+10 −10) AAA GAC UUA CCU UAA GAU AC167 H44A(−06 +14) AUC UGU CAA AUC GCC UGC AG 168 H46D(+16 −04)UUA CCU UGA CUU GCU CAA GC 169 H46A(+90 +109) UCC AGG UUC AAG UGG GAU AC170 H47A(+76 +100) GCU CUU CUG GGC UUA UGG GAG CAC U 171 H47D(+25 −02)ACC UUU AUC CAC UGG AGA UUU GUC UGC 172 H47A(−9 +12)UUC CAC CAG UAA CUG AAA CAG 173 H50A(+02 +30)CCA CUC AGA GCU CAG AUC UUC UAA CUU CC 174 H50A(+07 +33)CUU CCA CUC AGA GCU CAG AUC UUC UAA 175 H50D(+07 −18)GGG AUC CAG UAU ACU UAC AGG CUC C 176 H51A(−01 +25)ACC AGA GUA ACA GUC UGA GUA GGA GC 177 H51D(+16 −07)CUC AUA CCU UCU GCU UGA UGA UC 178 H51A(+111 +134)UUC UGU CCA AGC CCG GUU GAA AUC 179 H51A(+61 +90)ACA UCA AGG AAG AUG GCA UUU CUA GUU UGG 180 H51A(+66 +90)ACA UCA AGG AAG AUG GCA UUU CUA G 181 H51A(+66 +95)CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG 182 H51D(+08 −17)AUC AUU UUU UCU CAU ACC UUC UGC U 183 H51A/D(+08 −17) &AUC AUU UUU UCU CAU ACC UUC UGC UAG (−15+) GAG CUA AAA 184H51A(+175 +195) CAC CCA CCA UCA CCC UCU GUG 185 H51A(+199 +220)AUC AUC UCG UUG AUA UCC UCA A 186 H52A(−07 +14)UCC UGC AUU GUU GCC UGU AAG 187 H52A(+12 +41)UCC AAC UGG GGA CGC CUC UGU UCC AAA UCC 188 H52A(+17 +37)ACU GGG GAC GCC UCU GUU CCA 189 H52A(+93 +112)CCG UAA UGA UUG UUC UAG CC 190 H52D(+05 −15) UGU UAA AAA ACU UAC UUC GA191 H53A(+45 +69) CAU UCA ACU GUU GCC UCC GGU UCU G 192 H53A(+39 +62)CUG UUG CCU CCG GUU CUG AAG GUG 193 H53A(+39 +69)CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G 194 H53D(+14 −07)UAC UAA CCU UGG UUU CUG UGA 195 H53A(+23 +47)CUG AAG GUG UUC UUG UAC UUC AUC C 196 H53A(+150 +176)UGU AUA GGG ACC CUC CUU CCA UGA CUC 197 H53D(+20 −05)CUA ACC UUG GUU UCU GUG AUU UUC U 198 H53D(+09 −18)GGU AUC UUU GAU ACU AAC CUU GGU UUC 199 H53A(−12 +10)AUU CUU UCA ACU AGA AUA AAA G 200 H53A(−07 +18)GAU UCU GAA UUC UUU CAA CUA GAA U 201 H53A(+07 +26)AUC CCA CUG AUU CUG AAU UC 202 H53A(+124 +145)UUG GCU CUG GCC UGU CCU AAG A 203 H46A(+86 +115)CUC UUU UCC AGG UUC AAG UGG GAU ACU AGC 204 H46A(+107 +137)CAA GCU UUU CUU UUA GUU GCU GCU CUU UUC C 205 H46A(−10 +20)UAU UCU UUU GUU CUU CUA GCC UGG AGA AAG 206 H46A(+50 +77)CUG CUU CCU CCA ACC AUA AAA CAA AUU C 207 H45A(−06 +20)CCA AUG CCA UCC UGG AGU UCC UGU AA 208 H45A(+91 +110)UCC UGU AGA AUA CUG GCA UC 209 H45A(+125 +151)UGC AGA CCU CCU GCC ACC GCA GAU UCA 210 H45D(+16 −04)CUA CCU CUU UUU UCU GUC UG 211 H45A(+71 +90) UGU UUU UGA GGA UUG CUG AA

TABLE 1BDescription of a cocktail of 2′-O-methyl phosphorothioate antisenseoligonucleotides that have been used to date to study induced exonskipping during the processing of the dystrophin pre-mRNA. SEQ IDSEQUENCE NUCLEOTIDE SEQUENCE (5′-3′) 81 H20A(+44 +71)CUG GCA GAA UUC GAU CCA CCG GCU GUU C 82 H20A(+149 +170)CAG CAG UAG UUG UCA UCU GCU C 79 H19A(+35 +65)GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U 81 H20A(+44 +71)CUG GCA GAA UUC GAU CCA CCG GCU GUU C 82 H20A(+149 +170)CAG CAG UAG UUG UCA UCU GCU C 194 H53D(+14 −07)UAC UAA CCU UGG UUU CUG UGA 195 H53A(+23 +47)CTG AAG GUG UUC UUG UAC UUC AUC C 196 H53A(+150 +175)UGU AUA GGG ACC CUC CUU CCA UGA CUC

TABLE 1C Description of a “weasel”of 2′-O-methyl phosphorothioate antisense oligonucleotides that have been used to date to study induced exonskipping during the processing of the dystrophin pre-mRNA. SEQ IDSEQUENCE NUCELOTIDE SEQUENCE (5′-3′)  80 H20A(+44 +71)-CUG GCA GAA UUC GAU CCA CCG GCU GUU C-  82 H20A(+149 +170)CAG CAG UAG UUG UCA UCU GCU C  81 H19A(+35 +53)-GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U  88 H20A(+44 +63)--AUU CGA UCU ACC GGC UGU UC-  89 H20A(+149 +168)AA CUG CUG GCA UCU UGC AGU U  80 H19A(+35 +53)-GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U  88 H20A(+44 +63)-AUU CGA UCU ACC GGC UGU UC-  80 H19A(+35 +53)-GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U  89 H20A(+149 +168)-AA CUG CUG GCA UCU UGC AGU U 138 H34A(+46 +70)-CAU UCA UUU CCU UUC GCA UCU UAC G- 139 H34A(+94 +120)UGA UCU CUU UGU CAA UUC CAU AUC UG 124 H31D(+03 −22)-UU-UAG UUU CUG AAA UAA CAU AUA CCU G-UU- 144 H35A(+24 +43)UCU UCA GGU GCA CCU UCU GU 195 H53A(+23 +47)-AA-CUG AAG GUG UUC UUG UAC UUC AUC C- 196 H53A(+150 +175)-AA-UGU AUA GGG ACC CUC CUU CCA UGA CUC-AA 194 H53D(+14 −07)UAC UAA CCU UGG UUU CUG UGA — Aimed at exonsCAG CAG UAG UUG UCA UCU GCU CAA CUG 19/20/20GCA GAA UUC GAU CCA CCG GCU GUU CAA GCC UGA GCU GAU CUG CUC GCA UCUUGC AGU

DETAILED DESCRIPTION OF THE INVENTION

General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variation and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

Sequence identity numbers (SEQ ID NO:) containing nucleotide and aminoacid sequence information included in this specification are collectedat the end of the description and have been prepared using the programmePatentIn Version 3.0. Each nucleotide or amino acid sequence isidentified in the sequence listing by the numeric indicator <210>followed by the sequence identifier (e.g. <210>1, <210>2, etc.). Thelength, type of sequence and source organism for each nucleotide oramino acid sequence are indicated by information provided in the numericindicator fields <211>, <212> and <213>, respectively. Nucleotide andamino acid sequences referred to in the specification are defined by theinformation provided in numeric indicator field <400> followed by thesequence identifier (e.g. <400>1, <400>2, etc.).

An antisense molecules nomenclature system was proposed and published todistinguish between the different antisense molecules (see Mann et al.,(2002) J Gen Med 4, 644-654). This nomenclature became especiallyrelevant when testing several slightly different antisense molecules,all directed at the same target region, as shown below:

H # A/D (x:y).

The first letter designates the species (e.g. H: human, M: murine, C:canine)

“#” designates target dystrophin exon number.

“A/D” indicates acceptor or donor splice site at the beginning and endof the exon, respectively.

(x y) represents the annealing coordinates where “−” or “+” indicateintronic or exonic sequences respectively. As an example, A(−6+18) wouldindicate the last 6 bases of the intron preceding the target exon andthe first 18 bases of the target exon. The closest splice site would bethe acceptor so these coordinates would be preceded with an “A”.Describing annealing coordinates at the donor splice site could beD(+2−18) where the last 2 exonic bases and the first 18 intronic basescorrespond to the annealing site of the antisense molecule. Entirelyexonic annealing coordinates that would be represented by A(+65+85),that is the site between the 65^(th) and 85^(th) nucleotide from thestart of that exon.

The entire disclosures of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference. Noadmission is made that any of the references constitute prior art or arepart of the common general knowledge of those working in the field towhich this invention relates.

As used necessarily herein the term “derived” and “derived from” shallbe taken to indicate that a specific integer may be obtained from aparticular source albeit not directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

Description of the Preferred Embodiment

When antisense molecule(s) are targeted to nucleotide sequences involvedin splicing in exons within pre-mRNA sequences, normal splicing of theexon may be inhibited causing the splicing machinery to by-pass theentire mutated exon from the mature mRNA. The concept of antisenseoligonucleotide induced exon skipping is shown in FIG. 2. In many genes,deletion of an entire exon would lead to the production of anon-functional protein through the loss of important functional domainsor the disruption of the reading frame. In some proteins, however, it ispossible to shorten the protein by deleting one or more exons, withoutdisrupting the reading frame, from within the protein without seriouslyaltering the biological activity of the protein. Typically, suchproteins have a structural role and or possess functional domains attheir ends. The present invention describes antisense molecules capableof binding to specified dystrophin pre-mRNA targets and re-directingprocessing of that gene.

Antisense Molecules

According to a first aspect of the invention, there is providedantisense molecules capable of binding to a selected target to induceexon skipping. To induce exon skipping in exons of the Dystrophin genetranscript, the antisense molecules are preferably selected from thegroup of compounds shown in Table 1A. There is also provided acombination or “cocktail” of two or more antisense oligonucleotidescapable of binding to a selected target to induce exon skipping. Toinduce exon skipping in exons of the Dystrophin gene transcript, theantisense molecules in a “cocktail” are preferably selected from thegroup of compounds shown in Table 1B. Alternatively, exon skipping maybe induced by antisense oligonucleotides joined together “weasels”preferably selected from the group of compounds shown in Table 1C.

Designing antisense molecules to completely mask consensus splice sitesmay not necessarily generate any skipping of the targeted exon.Furthermore, the inventors have discovered that size or length of theantisense oligonucleotide itself is not always a primary factor whendesigning antisense molecules. With some targets such as exon 19,antisense oligonucleotides as short as 12 bases were able to induce exonskipping, albeit not as efficiently as longer (20-31 bases)oligonucleotides. In some other targets, such as murine dystrophin exon23, antisense oligonucleotides only 17 residues long were able to inducemore efficient skipping than another overlapping compound of 25nucleotides.

The inventors have also discovered that there does not appear to be anystandard motif that can be blocked or masked by antisense molecules toredirect splicing. In some exons, such as mouse dystrophin exon 23, thedonor splice site was the most amenable to target to re-direct skippingof that exon. It should be noted that designing and testing a series ofexon 23 specific antisense molecules to anneal to overlapping regions ofthe donor splice site showed considerable variation in the efficacy ofinduced exon skipping. As reported in Mann et al., (2002) there was asignificant variation in the efficiency of bypassing the nonsensemutation depending upon antisense oligonucleotide annealing (“Improvedantisense oligonucleotide induced exon skipping in the mdx mouse modelof muscular dystrophy”. J Gen Med 4: 644-654). Targeting the acceptorsite of exon 23 or several internal domains was not found to induce anyconsistent exon 23 skipping.

In other exons targeted for removal, masking the donor splice site didnot induce any exon skipping. However, by directing antisense moleculesto the acceptor splice site (human exon 8 as discussed below), strongand sustained exon skipping was induced. It should be noted that removalof human exon 8 was tightly linked with the co-removal of exon 9. Thereis no strong sequence homology between the exon 8 antisenseoligonucleotides and corresponding regions of exon 9 so it does notappear to be a matter of cross reaction. Rather the splicing of thesetwo exons is inextricably linked. This is not an isolated instance asthe same effect is observed in canine cells where targeting exon 8 forremoval also resulted in the skipping of exon 9. Targeting exon 23 forremoval in the mouse dystrophin pre-mRNA also results in the frequentremoval of exon 22 as well. This effect occurs in a dose dependentmanner and also indicates close coordinated processing of 2 adjacentexons.

In other targeted exons, antisense molecules directed at the donor oracceptor splice sites did not induce exon skipping while annealingantisense molecules to intra-exonic regions (i.e. exon splicingenhancers within human dystrophin exon 6) was most efficient at inducingexon skipping. Some exons, both mouse and human exon 19 for example, arereadily skipped by targeting antisense molecules to a variety of motifs.That is, targeted exon skipping is induced after using antisenseoligonucleotides to mask donor and acceptor splice sites or exonsplicing enhancers.

To identify and select antisense oligonucleotides suitable for use inthe modulation of exon skipping, a nucleic acid sequence whose functionis to be modulated must first be identified. This may be, for example, agene (or mRNA transcribed form the gene) whose expression is associatedwith a particular disorder or disease state, or a nucleic acid moleculefrom an infectious agent. Within the context of the present invention,preferred target site(s) are those involved in mRNA splicing (i.e.splice donor sites, splice acceptor sites, or exonic splicing enhancerelements). Splicing branch points and exon recognition sequences orsplice enhancers are also potential target sites for modulation of mRNAsplicing.

Preferably, the present invention aims to provide antisense moleculescapable of binding to a selected target in the dystrophin pre-mRNA toinduce efficient and consistent exon skipping. Duchenne musculardystrophy arises from mutations that preclude the synthesis of afunctional dystrophin gene product. These Duchenne muscular dystrophygene defects are typically nonsense mutations or genomic rearrangementssuch as deletions, duplications or micro-deletions or insertions thatdisrupt the reading frame. As the human dystrophin gene is a large andcomplex gene with the 79 exons being spliced together to generate amature mRNA with an open reading frame of approximately 11,000 bases,there are many positions where these mutations can occur. Consequently,a comprehensive antisense oligonucleotide based therapy to address manyof the different disease-causing mutations in the dystrophin gene willrequire that many exons can be targeted for removal during the splicingprocess.

Within the context of the present invention, preferred target site(s)are those involved in mRNA splicing (i.e. splice donor sites, spliceacceptor sites or exonic splicing enhancer elements). Splicing branchpoints and exon recognition sequences or splice enhancers are alsopotential target sites for modulation of mRNA splicing.

The oligonucleotide and the DNA or RNA are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridisable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity or precise pairingsuch that stable and specific binding occurs between the oligonucleotideand the DNA or RNA target. It is understood in the art that the sequenceof an antisense molecule need not be 100% complementary to that of itstarget sequence to be specifically hybridisable. An antisense moleculeis specifically hybridisable when binding of the compound to the targetDNA or RNA molecule interferes with the normal function of the targetDNA or RNA to cause a loss of utility, and there is a sufficient degreeof complementarity to avoid non-specific binding of the antisensecompound to non-target sequences under conditions in which specificbinding is desired, i.e., under physiological conditions in the case ofin vivo assays or therapeutic treatment, and in the case of in vitroassays, under conditions in which the assays are performed.

While the above method may be used to select antisense molecules capableof deleting any exon from within a protein that is capable of beingshortened without affecting its biological function, the exon deletionshould not lead to a reading frame shift in the shortened transcribedmRNA. Thus, if in a linear sequence of three exons the end of the firstexon encodes two of three nucleotides in a codon and the next exon isdeleted then the third exon in the linear sequence must start with asingle nucleotide that is capable of completing the nucleotide tripletfor a codon. If the third exon does not commence with a singlenucleotide there will be a reading frame shift that would lead to thegeneration of truncated or a non-functional protein.

It will be appreciated that the codon arrangements at the end of exonsin structural proteins may not always break at the end of a codon,consequently there may be a need to delete more than one exon from thepre-mRNA to ensure in-frame reading of the mRNA. In such circumstances,a plurality of antisense oligonucleotides may need to be selected by themethod of the invention wherein each is directed to a different regionresponsible for inducing splicing in the exons that are to be deleted.

The length of an antisense molecule may vary so long as it is capable ofbinding selectively to the intended location within the pre-mRNAmolecule. The length of such sequences can be determined in accordancewith selection procedures described herein. Generally, the antisensemolecule will be from about 10 nucleotides in length up to about 50nucleotides in length. It will be appreciated however that any length ofnucleotides within this range may be used in the method. Preferably, thelength of the antisense molecule is between 17 to 30 nucleotides inlength.

In order to determine which exons can be connected in a dystrophin gene,reference should be made to an exon boundary map. Connection of one exonwith another is based on the exons possessing the same number at the 3′border as is present at the 5′ border of the exon to which it is beingconnected. Therefore, if exon 7 were deleted, exon 6 must connect toeither exons 12 or 18 to maintain the reading frame. Thus, antisenseoligonucleotides would need to be selected which redirected splicing forexons 7 to 11 in the first instance or exons 7 to 17 in the secondinstance. Another and somewhat simpler approach to restore the readingframe around an exon 7 deletion would be to remove the two flankingexons. Induction of exons 6 and 8 skipping should result in an in-frametranscript with the splicing of exons 5 to 9. In practice however,targeting exon 8 for removal from the pre-mRNA results in the co-removalof exon 9 so the resultant transcript would have exon 5 joined to exon10. The inclusion or exclusion of exon 9 does not alter the readingframe. Once the antisense molecules to be tested have been identified,they are prepared according to standard techniques known in the art. Themost common method for producing antisense molecules is the methylationof the 2′ hydroxyribose position and the incorporation of aphosphorothioate backbone produces molecules that superficially resembleRNA but that are much more resistant to nuclease degradation.

To avoid degradation of pre-mRNA during duplex formation with theantisense molecules, the antisense molecules used in the method may beadapted to minimise or prevent cleavage by endogenous RNase H. Thisproperty is highly preferred as the treatment of the RNA with theunmethylated oligonucleotides either intracellularly or in crudeextracts that contain RNase H leads to degradation of the pre-mRNA:antisense oligonucleotide duplexes. Any form of modified antisensemolecules that is capable of by-passing or not inducing such degradationmay be used in the present method. An example of antisense moleculeswhich when duplexed with RNA are not cleaved by cellular RNase H is2′-O-methyl derivatives. 2′-O-methyl-oligoribonucleotides are verystable in a cellular environment and in animal tissues, and theirduplexes with RNA have higher Tm values than their ribo- ordeoxyribo-counterparts.

Antisense molecules that do not activate RNase H can be made inaccordance with known techniques (see, e.g., U.S. Pat. No. 5,149,797).Such antisense molecules, which may be deoxyribonucleotide orribonucleotide sequences, simply contain any structural modificationwhich sterically hinders or prevents binding of RNase H to a duplexmolecule containing the oligonucleotide as one member thereof, whichstructural modification does not substantially hinder or disrupt duplexformation. Because the portions of the oligonucleotide involved induplex formation are substantially different from those portionsinvolved in RNase H binding thereto, numerous antisense molecules thatdo not activate RNase H are available. For example, such antisensemolecules may be oligonucleotides wherein at least one, or all, of theinter-nucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphorothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues may be modified as described. In another non-limiting example,such antisense molecules are molecules wherein at least one, or all, ofthe nucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄, linear orbranched, saturated or unsaturated alkyl, such as methyl, ethyl,ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example,every other one of the nucleotides may be modified as described.

While antisense oligonucleotides are a preferred form of the antisensemolecules, the present invention comprehends other oligomeric antisensemolecules, including but not limited to oligonucleotide mimetics such asare described below.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural inter-nucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their inter-nucleoside backbone can also beconsidered to be oligonucleosides.

In other preferred oligonucleotide mimetics, both the sugar and theinter-nucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleo-bases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Oligonucleotides may also include nucleobase (often referredto in the art simply as “base”) modifications or substitutions. Certainnucleo-bases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense molecules, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the increasedresistance to nuclease degradation, increased cellular uptake, and anadditional region for increased binding affinity for the target nucleicacid.

Methods of Manufacturing Antisense Molecules

The antisense molecules used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). One method for synthesising oligonucleotides on a modifiedsolid support is described in U.S. Pat. No. 4,458,066.

Any other means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives. In one such automated embodiment, diethyl-phosphoramiditesare used as starting materials and may be synthesized as described byBeaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

The antisense molecules of the invention are synthesised in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The molecules of the invention may also be mixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.

Therapeutic Agents

The present invention also can be used as a prophylactic or therapeutic,which may be utilised for the purpose of treatment of a genetic disease.

Accordingly, in one embodiment the present invention provides antisensemolecules that bind to a selected target in the dystrophin pre-mRNA toinduce efficient and consistent exon skipping described herein in atherapeutically effective amount admixed with a pharmaceuticallyacceptable carrier, diluent, or excipient.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similarly untoward reaction, such as gastricupset and the like, when administered to a patient. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thecompound is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Martin, Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa., (1990).

In a more specific form of the invention there are providedpharmaceutical compositions comprising therapeutically effective amountsof an antisense molecule together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents of various buffer content(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength andadditives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). The material may beincorporated into particulate preparations of polymeric compounds suchas polylactic acid, polyglycolic acid, etc. or into liposomes.Hylauronic acid may also be used. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, e.g., Martin,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 that are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilised form.

It will be appreciated that pharmaceutical compositions providedaccording to the present invention may be administered by any meansknown in the art. Preferably, the pharmaceutical compositions foradministration are administered by injection, orally, or by thepulmonary, or nasal route. The antisense molecules are more preferablydelivered by intravenous, intra-arterial, intraperitoneal,intramuscular, or subcutaneous routes of administration.

Antisense Molecule Based Therapy

Also addressed by the present invention is the use of antisensemolecules of the present invention, for manufacture of a medicament formodulation of a genetic disease.

The delivery of a therapeutically useful amount of antisense moleculesmay be achieved by methods previously published. For example,intracellular delivery of the antisense molecule may be via acomposition comprising an admixture of the antisense molecule and aneffective amount of a block copolymer. An example of this method isdescribed in US patent application US 20040248833.

Other methods of delivery of antisense molecules to the nucleus aredescribed in Mann C J et al., (2001) [“Antisense-induced exon skippingand the synthesis of dystrophin in the mdx mouse”. Proc., Natl. Acad.Science, 98(1) 42-47] and in Gebski et al., (2003). Human MolecularGenetics, 12(15): 1801-1811.

A method for introducing a nucleic acid molecule into a cell by way ofan expression vector either as naked DNA or complexed to lipid carriers,is described in US patent U.S. Pat. No. 6,806,084.

It may be desirable to deliver the antisense molecule in a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes or liposome formulations.

Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. These formulations may have net cationic,anionic or neutral charge characteristics and are useful characteristicswith in vitro, in vivo and ex vivo delivery methods. It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-4.0.PHI.m can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, and DNA can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).

In order for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of theantisense molecule of interest at high efficiency while not compromisingtheir biological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Alternatively, the antisense construct may be combined with otherpharmaceutically acceptable carriers or diluents to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular, oral or transdermaladministration.

The routes of administration described are intended only as a guidesince a skilled practitioner will be able to determine readily theoptimum route of administration and any dosage for any particular animaland condition. Multiple approaches for introducing functional newgenetic material into cells, both in vitro and in vivo have beenattempted (Friedmann (1989) Science, 244:1275-1280). These approachesinclude integration of the gene to be expressed into modifiedretroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research51(18), suppl.: 5074S-5079S); integration into non-retrovirus vectors(Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991)Science, 252:431-434); or delivery of a transgene linked to aheterologous promoter-enhancer element via liposomes (Friedmann (1989),supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, etal. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp.Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl.Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific,cation-based transport systems (Wu and Wu (1988) J. Biol. Chem.,263:14621-14624) or the use of naked DNA, expression vectors (Nabel etal. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Directinjection of transgenes into tissue produces only localized expression(Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra; Brigham et al.(1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). TheBrigham et al. group (Am. J. Med. Sci. (1989) 298:278-281 and ClinicalResearch (1991) 39 (abstract)) have reported in vivo transfection onlyof lungs of mice following either intravenous or intratrachealadministration of a DNA liposome complex. An example of a review articleof human gene therapy procedures is: Anderson, Science (1992)256:808-813.

The antisense molecules of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such pro-drugs, andother bioequivalents.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including rectal delivery), pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, (including by nebulizer,intratracheal, intranasal, epidermal and transdermal), oral orparenteral. Parenteral administration includes intravenous,intra-arterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Kits of the Invention

The invention also provides kits for treatment of a patient with agenetic disease which kit comprises at least an antisense molecule,packaged in a suitable container, together with instructions for itsuse.

In a preferred embodiment, the kits will contain at least one antisensemolecule as shown in Table 1A, or a cocktail of antisense molecules asshown in Table 1B or a “weasel” compound as shown in Table 1C. The kitsmay also contain peripheral reagents such as buffers, stabilizers, etc.

Those of ordinary skill in the field should appreciate that applicationsof the above method has wide application for identifying antisensemolecules suitable for use in the treatment of many other diseases.

EXAMPLES

The following Examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these Examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.The references cited herein are expressly incorporated by reference.

Methods of molecular cloning, immunology and protein chemistry, whichare not explicitly described in the following examples, are reported inthe literature and are known by those skilled in the art. General textsthat described conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art, included, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II,MRL Press, Ltd., Oxford, U.K. (1985); and Ausubel, F., Brent, R.,Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K.Current Protocols in Molecular Biology. Greene PublishingAssociates/Wiley Intersciences, New York (2002).

Determining Induced Exon Skipping in Human Muscle Cells

Attempts by the inventors to develop a rational approach in antisensemolecules design were not completely successful as there did not appearto be a consistent trend that could be applied to all exons. As such,the identification of the most effective and therefore most therapeuticantisense molecules compounds has been the result of empirical studies.

These empirical studies involved the use of computer programs toidentify motifs potentially involved in the splicing process. Othercomputer programs were also used to identify regions of the pre-mRNAwhich may not have had extensive secondary structure and thereforepotential sites for annealing of antisense molecules. Neither of theseapproaches proved completely reliable in designing antisenseoligonucleotides for reliable and efficient induction of exon skipping.

Annealing sites on the human dystrophin pre-mRNA were selected forexamination, initially based upon known or predicted motifs or regionsinvolved in splicing. 2OMe antisense oligonucleotides were designed tobe complementary to the target sequences under investigation and weresynthesised on an Expedite 8909 Nucleic Acid Synthesiser. Uponcompletion of synthesis, the oligonucleotides were cleaved from thesupport column and de-protected in ammonium hydroxide before beingdesalted. The quality of the oligonucleotide synthesis was monitored bythe intensity of the trityl signals upon each deprotection step duringthe synthesis as detected in the synthesis log. The concentration of theantisense oligonucleotide was estimated by measuring the absorbance of adiluted aliquot at 260 nm.

Specified amounts of the antisense molecules were then tested for theirability to induce exon skipping in an in vitro assay, as describedbelow.

Briefly, normal primary myoblast cultures were prepared from humanmuscle biopsies obtained after informed consent. The cells werepropagated and allowed to differentiate into myotubes using standardculturing techniques. The cells were then transfected with the antisenseoligonucleotides by delivery of the oligonucleotides to the cells ascationic lipoplexes, mixtures of antisense molecules or cationicliposome preparations.

The cells were then allowed to grow for another 24 hours, after whichtotal RNA was extracted and molecular analysis commenced. Reversetranscriptase amplification (RT-PCR) was undertaken to study thetargeted regions of the dystrophin pre-mRNA or induced exonicre-arrangements.

For example, in the testing of an antisense molecule for inducing exon19 skipping the RT-PCR test scanned several exons to detect involvementof any adjacent exons. For example, when inducing skipping of exon 19,RT-PCR was carried out with primers that amplified across exons 17 and21. Amplifications of even larger products in this area (i.e. exons13-26) were also carried out to ensure that there was minimalamplification bias for the shorter induced skipped transcript. Shorteror exon skipped products tend to be amplified more efficiently and maybias the estimated of the normal and induced transcript.

The sizes of the amplification reaction products were estimated on anagarose gel and compared against appropriate size standards. The finalconfirmation of identity of these products was carried out by direct DNAsequencing to establish that the correct or expected exon junctions havebeen maintained.

Once efficient exon skipping had been induced with one antisensemolecule, subsequent overlapping antisense molecules may be synthesizedand then evaluated in the assay as described above. Our definition of anefficient antisense molecule is one that induces strong and sustainedexon skipping at transfection concentrations in the order of 300 nM orless.

Antisense Oligonucleotides Directed at Exon 8

Antisense oligonucleotides directed at exon 8 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 3 shows differing efficiencies of two antisense molecules directedat exon 8 acceptor splice site. H8A(−06+18) [SEQ ID NO:1], which annealsto the last 6 bases of intron 7 and the first 18 bases of exon 8,induces substantial exon 8 and 9 skipping when delivered into cells at aconcentration of 20 nM. The shorter antisense molecule, H8A(−06+14) [SEQID NO: 4] was only able to induce exon 8 and 9 skipping at 300 nM, aconcentration some 15 fold higher than H8A(−06+18), which is thepreferred antisense molecule.

This data shows that some particular antisense molecules induceefficient exon skipping while another antisense molecule, which targetsa near-by or overlapping region, can be much less efficient. Titrationstudies show one compound is able to induce targeted exon skipping at 20nM while the less efficient antisense molecules only induced exonskipping at concentrations of 300 nM and above. Therefore, we have shownthat targeting of the antisense molecules to motifs involved in thesplicing process plays a crucial role in the overall efficacy of thatcompound.

Efficacy refers to the ability to induce consistent skipping of a targetexon. However, sometimes skipping of the target exons is consistentlyassociated with a flanking exon. That is, we have found that thesplicing of some exons is tightly linked. For example, in targeting exon23 in the mouse model of muscular dystrophy with antisense moleculesdirected at the donor site of that exon, dystrophin transcripts missingexons 22 and 23 are frequently detected. As another example, when usingan antisense molecule directed to exon 8 of the human dystrophin gene,all induced transcripts are missing both exons 8 and 9. Dystrophintranscripts missing only exon 8 are not observed.

Table 2 below discloses antisense molecule sequences that induce exon 8(and 9) skipping.

TABLE 2 Antisense Oligonucleotide Ability to induce name Sequenceskipping H8A(−06 +18) 5′-GAU AGG UGG UAU CAA CAU CUG UAAVery strong to 20 nM H8A(−03 +18) 5′-GAU AGG UGG UAU CAA CAU CUGVery strong skipping to 40 nM H8A(−07 +18)5′-GAU AGG UGG UAU CAA CAU CUG UAA G Strong skipping to 40 nMH8A(−06 +14) 5′-GGU GGU AUC AAC AUC UGU AA Skipping to 300 nMH8A(−10 +10) 5′-GUA UCA ACA UCU GUA AGC AC Patchy/weak skippingto 100 nm

Antisense Oligonucleotides Directed at Exon 7

Antisense oligonucleotides directed at exon 7 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 4 shows the preferred antisense molecule, H7A(+45+67) [SEQ ID NO:6], and another antisense molecule, H7A(+2+26) [SEQ ID NO: 7], inducingexon 7 skipping. Nested amplification products span exons 3 to 9.Additional products above the induced transcript missing exon 7 arisefrom amplification from carry-over outer primers from the RT-PCR as wellas heteroduplex formation.

Table 3 below discloses antisense molecule sequences for induced exon 7skipping.

TABLE 3 Antisense Oligonucleotide Ability to induce name Sequenceskipping H7A(+45 +67) 5′-UGC AUG UUC CAG UCG UUG UGU GGStrong skipping to 20 nM H7A(+02 +26)5′-CAC UAU UCC AGU CAA AUA GGU CUG G Weak skipping at 100 nMH7D(+15 −10) 5′-AUU UAC CAA CCU UCA GGA UCG AGU A Weak skipping to300 nM Weak skipping to H7A(−18 +03) 5′-GGC CUA AAA CAC AUA CAC AUA300 nM

Antisense Oligonucleotides Directed at Exon 6

Antisense oligonucleotides directed at exon 6 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 5 shows an example of two non-preferred antisense moleculesinducing very low levels of exon 6 skipping in cultured human cells.Targeting this exon for specific removal was first undertaken during astudy of the canine model using the oligonucleotides as listed in Table4, below. Some of the human specific oligonucleotides were alsoevaluated, as shown in FIG. 5. In this example, both antisense moleculestarget the donor splice site and only induced low levels of exon 6skipping. Both H6D(+4−21) [SEQ ID NO: 17] and H6D(+18−4) [SEQ ID NO: 18]would be regarded as non-preferred antisense molecules.

One antisense oligonucleotide that induced very efficient exon 6skipping in the canine model, C6A(+69+91) [SEQ ID NO: 14], would annealperfectly to the corresponding region in human dystrophin exon 6. Thiscompound was evaluated, found to be highly efficient at inducingskipping of that target exon, as shown in FIG. 6 and is regarded as thepreferred compound for induced exon 6 skipping. Table 4 below disclosesantisense molecule sequences for induced exon 6 skipping.

TABLE 4 Antisense Oligo Ability to induce name Sequence skippingC6A(−10 +10) 5′ CAU UUU UGA CCU ACA UGU GG No skipping C6A(−14 +06) 5′UUU GAC CUA CAU GUG GAA AG No skipping C6A(−14 +12) 5′UAC AUU UUU GAC CUA CAU GUG No skipping GAA AG C6A(−13 +09) 5′AUU UUU GAC CUA CAU GGG AAA G No skipping CH6A(+69 +91) 5′UAC GAG UUG AUU GUC GGA CCC Strong skipping to AG 20 nM C6D(+12 −13) 5′GUG GUC UCC UUA CCU AUG ACU Weak skipping at GUG G 300 nM C6D(+06 −11)5′ GGU CUC CUU ACC UAU GA No skipping H6D(+04 −21) 5′UGU CUC AGU AAU CUU CUU ACC Weak skipping to UAU 50 nM H6D(+18 −04) 5′UCU UAC CUA UGA CUA UGG AUG Very weak skipping AGA to 300 nM

Antisense Oligonucleotides Directed at Exon 4

Antisense oligonucleotides directed at exon 4 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 7 shows an example of a preferred antisense molecule inducingskipping of exon 4 skipping in cultured human cells. In this example,one preferred antisense compound, H4A(+13+32) [SEQ ID NO:19], whichtargeted a presumed exonic splicing enhancer induced efficient exonskipping at a concentration of 20 nM while other non-preferred antisenseoligonucleotides failed to induce even low levels of exon 4 skipping.Another preferred antisense molecule inducing skipping of exon 4 wasH4A(+111+40) [SEQ ID NO:22], which induced efficient exon skipping at aconcentration of 20 nM.

Table 5 below discloses antisense molecule sequences for inducing exon 4skipping.

TABLE 5 Antisense Oligonucleotide Ability to induce name Sequenceskipping H4A(+13 +32) 5′ GCA UGA ACU CUU GUG GAU CC Skipping to 20 nMH4A(+11 +40) 5′ UGU UCA GGG CAU GAA CUC UUG UGG AUC Skipping to 20 nMCUU H4D(+04 −16) 5′ CCA GGG UAC UAC UUA CAU UA No skipping H4D(−24 −44)5′ AUC GUG UGU CAC AGC AUC CAG No skipping

Antisense Oligonucleotides Directed at Exon 3

Antisense oligonucleotides directed at exon 3 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H3A(+30+60) [SEQ ID NO:23] induced substantial exon 3 skipping whendelivered into cells at a concentration of 20 nM to 600 nM. Theantisense molecule, H3A(+35+65) [SEQ ID NO: 24] induced exon skipping at300 nM.

Table 6 below discloses antisense molecule sequences that induce exon 3skipping.

TABLE 6 Antisense Oligonucleotide Ability to induce name Sequenceskipping H3A(+30 +60) UAG GAG GCG CCU CCC AUC CUG UAG GUCModerate skipping ACU G to 20 to 600 nM H3A(+35 +65)AGG UCU AGG AGG CGC CUC CCA UCC UGU Working to 300 nM AGG U H3A(+30 +54)GCG CCU CCC AUC CUG UAG GUC ACU G Moderate 100-600 nM H3D(+46 −21)CUU CGA GGA GGU CUA GGA GGC GCC UC No skipping H3A(+30 +50)CUC CCA UCC UGU AGG UCA CUG Moderate 20-600 nM H3D(+19 −03)UAC CAG UUU UUG CCC UGU CAG G No skipping H3A(−06 +20)UCA AUA UGC UGC UUCCCA AAC UGA AA No skipping H3A(+37 +61)CUA GGA GGC GCC UCC CAU CCU GUA G No skipping

Antisense Oligonucleotides Directed at Exon 5

Antisense oligonucleotides directed at exon 5 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H5A(+20+50) [SEQ ID NO:31] induces substantial exon 5 skipping whendelivered into cells at a concentration of 100 nM. Table 7 below showsother antisense molecules tested. The majority of these antisensemolecules were not as effective at exon skipping as H5A(+20+50).However, H5A(+15+45) [SEQ ID NO: 40] was able to induce exon 5 skippingat 300 nM.

Table 7 below discloses antisense molecule sequences that induce exon 5skipping.

TABLE 7 Antisense Oligonucleotide Ability to induce name Sequenceskipping H5A(+20 +50) UUA UGA UUU CCA UCU ACG AUG UCA GUAWorking to 100 nM CUU C H5D(+25 −05) CUU ACC UGC CAG UGG AGG AUU AUA UUCNo skipping CAA A H5D(+10 −15) CAU CAG GAU UCU UAC CUG CCA GUG GInconsistent at 300 nM H5A(+10 +34) CGA UGU CAG UAC UUC CAA UAU UCA CVery weak H5D(−04 −21) ACC AUU CAU CAG GAU UCU No skipping H5D(+16 −02)ACC UGC CAG UGG AGG AUU No skipping H5A(−07 +20)CCA AUA UUC ACU AAA UCA ACC UGU UAA No skipping H5D(+18 −12)CAG GAU UCU UAC CUG CCA GUG GAG GAU No skipping UAU H5A(+05 +35)ACG AUG UCA GUA CUU CCA AUA UUC ACU No skipping AAA U H5A(+15 +45)AUU UCC AUC UAC GAU GUC AGU ACU UCC Working to 300 nM AAU A

Antisense Oligonucleotides Directed at Exon 10

Antisense oligonucleotides directed at exon 10 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H10A(−05+16) [SEQ ID NO:41] induced substantial exon 10 skipping whendelivered into cells. Table 8 below shows other antisense moleculestested. The antisense molecules ability to induce exon skipping wasvariable. Table 8 below discloses antisense molecule sequences thatinduce exon 10 skipping.

TABLE 8 Antisense Oligonucleotide Ability to induce name Sequenceskipping H10A(−05 +16) CAG GAG CUU CCA AAU GCU GCA Not testedH10A(−05 +24) CUU GUC UUC AGG AGC UUC CAA AUG CUG Not tested CAH10A(+98 +119) UCC UCA GCA GAA AGA AGC CAC G Not tested H10A(+130 +149)UUA GAA AUC UCU CCU UGU GC No skipping H10A(−33 −14)UAA AUU GGG UGU UAC ACA AU No skipping

Antisense Oligonucleotides Directed at Exon 11

Antisense oligonucleotides directed at exon 11 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 8B shows an example of H11A(+75+97) [SEQ ID NO:49] antisensemolecule inducing exon 11 skipping in cultured human cells. H11A(+75+97)induced substantial exon 11 skipping when delivered into cells at aconcentration of 5 nM. Table 9 below shows other antisense moleculestested. The antisense molecules ability to induce exon skipping wasobserved at 100 nM.

TABLE 9 Antisense Oligonucleotide Ability to induce name Sequenceskipping H11D(+26 +49) CCC UGA GGC AUU CCC AUC UUG AAUSkipping at 100 nM H11D(+11 −09) AGG ACU UAC UUG CUU UGU UUSkipping at 100 nM H11A(+118 +140) CUU GAA UUU AGG AGA UUC AUC UGSkipping at 100 nM H11A(+75 +97) CAU CUU CUG AUA AUU UUC CUG UUSkipping at 100 nM H11D(+26 +49) CCC UGA GGC AUU CCC AUC UUG AAUSkipping at 5 nM

Antisense Oligonucleotides Directed at Exon 12

Antisense oligonucleotides directed at exon 12 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H12A(+52+75) [SEQ ID NO:50] induced substantial exon 12 skipping whendelivered into cells at a concentration of 5 nM, as shown in FIG. 8A.Table 10 below shows other antisense molecules tested at a concentrationrange of 5, 25, 50, 100, 200 and 300 nM. The antisense molecules abilityto induce exon skipping was variable.

TABLE 10  Antisense Ability Oligonucleotide to induce name Sequenceskipping H12A(+52+75) UCU UCU GUU UUU GUU Skipping AGC CAG UCA at 5 nMH12A(−10+10) UCU AUG UAA ACU GAA Skipping AAU UU at 100 nM H12A(+11+30)UUC UGG AGA UCC AUU No skipping AAA AC

Antisense Oligonucleotides Directed at Exon 13

Antisense oligonucleotides directed at exon 13 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H13A(+77+100) [SEQ ID NO:53] induced substantial exon 13 skipping whendelivered into cells at a concentration of 5 nM. Table 11 below includestwo other antisense molecules tested at a concentration range of 5, 25,50, 100, 200 and 300 nM. These other antisense molecules were unable toinduce exon skipping.

TABLE 11  Antisense Ability Oligonucleotide to induce name Sequenceskipping H13A(+77+100) CAG CAG UUG CGU GAU CUC Skipping CAC UAG at 5 nMH13A(+55+75) UUC AUC AAC UAC CAC CAC No skipping CAU H13D(+06−19) CUA AGC AAA AUA AUC UGA No skipping CCU UAA G

Antisense Oligonucleotides Directed at Exon 14

Antisense oligonucleotides directed at exon 14 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H14A(+37+64) [SEQ ID NO:56] induced weak exon 14 skipping when deliveredinto cells at a concentration of 100 nM. Table 12 below includes otherantisense molecules tested at a concentration range of 5, 25, 50, 100,200 and 300 nM. The other antisense molecules were unable to induce exonskipping at any of the concentrations tested.

TABLE 12 Antisense Ability Oligonucleotide to induce name Sequenceskipping H14A(+37+64) CUU GUA AAA GAA CCC AGC GGU CUU CUG U Skippingat 100 nM H14A(+14+35) CAU CUA CAG AUG UUU GCC CAU C No skippingH14A(+51+73) GAA GGA UGU CUU GUA AAA GAA CC No skipping H14D(−02+18)ACC UGU UCU UCA GUA AGA CG No skipping H14D(+14−10)CAU GAC ACA CCU GUU CUU CAG UAA No skipping H14A(+61+80)CAU UUG AGA AGG AUG UCU UG No skipping H14A(−12+12)AUC UCC CAA UAC CUG GAG AAG AGA No skipping

Antisense Oligonucleotides Directed at Exon 15

Antisense oligonucleotides directed at exon 15 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H15A(−12+19) [SEQ ID NO:63] and H15A(+48+71) [SEQ ID NO:64] inducedsubstantial exon 15 skipping when delivered into cells at aconcentration of 10 Nm, as shown in FIG. 9A. Table 13 below includesother antisense molecules tested at a concentration range of 5, 25, 50,100, 200 and 300 Nm. These other antisense molecules were unable toinduce exon skipping at any of the concentrations tested.

TABLE 13  Antisense Ability Oligonucleotide to induce name Sequenceskipping H15A(−12+19) GCC AUG CAC UAA AAA GGC ACU GCA AGA Skipping CAU Uat 5 Nm H15A(+48+71) UCU UUA AAG CCA GUU GUG UGA AUC Skipping at 5 NmH15A(+08+28) UUU CUG AAA GCC AUG CAC UAA No skipping H15A(−12+19)GCC AUG CAC UAA AAA GGC ACU GCA AGA No skipping CAU U H15D(+17−08)GUA CAU ACG GCC AGU UUU UGA AGA C No skipping

Antisense Oligonucleotides Directed at Exon 16

Antisense oligonucleotides directed at exon 16 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H16A(−12+19) [SEQ ID NO:67] and H16A(−06+25) [SEQ ID NO:68] inducedsubstantial exon 16 skipping when delivered into cells at aconcentration of 10 nM, as shown in FIG. 9B. Table 14 below includesother antisense molecules tested. H16A(−06+19) [SEQ ID NO:69] andH16A(+87+109) [SEQ ID NO:70] were tested at a concentration range of 5,25, 50, 100, 200 and 300 nM. These two antisense molecules were able toinduce exon skipping at 25 nM and 100 nM, respectively. Additionalantisense molecules were tested at 100, 200 and 300 nM and did notresult in any exon skipping.

TABLE 14  Antisense Ability Oligonucleotide to induce name Sequenceskipping H16A(−12+19) CUA GAU CCG CUU UUA AAA CCU GUU AAA Skipping ACA Aat 5 nM H16A(−06+25) UCU UUU CUA GAU CCG CUU UUA AAA CCU Skipping GUU Aat 5 nM H16A(−06+19) CUA GAU CCG CUU UUA AAA CCU GUU A Skipping at 25 nMH16A(+87+109) CCG UCU UCU GGG UCA CUG ACU UA Skipping at 100 nMH16A(−07+19) CUA GAU CCG CUU UUA AAA CCU GUU AA No skipping H16A(−07+13)CCG CUU UUA AAA CCU GUU AA No skipping H16A(+12+37)UGG AUU GCU UUU UCU UUU CUA GAU CC No skipping H16A(+92+116)CAU GCU UCC GUC UUC UGG GUC ACU G No skipping H16A(+45+67)G AUC UUG UUU GAG UGA AUA CAG U No skipping H16A(+105+126)GUU AUC CAG CCA UGC UUC CGU C No skipping H16D(+05−20)UGA UAA UUG GUA UCA CUA ACC UGU G No skipping H16D(+12−11)GUA UCA CUA ACC UGU GCU GUA C No skipping

Antisense Oligonucleotides Directed at Exon 19

Antisense oligonucleotides directed at exon 19 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H19A(+35+65) [SEQ ID NO:79] induced substantial exon 19 skipping whendelivered into cells at a concentration of 10 nM. This antisensemolecule also showed very strong exon skipping at concentrations of 25,50, 100, 300 and 600 nM.

FIG. 10 illustrates exon 19 and 20 skipping using a “cocktail” ofantisense oligonucleotides, as tested using gel electrophoresis. It isinteresting to note that it was not easy to induce exon 20 skippingusing single antisense oligonucleotides H20A(+44+71) [SEQ ID NO:81] orH20A(+149+170) [SEQ ID NO:82], as illustrated in sections 2 and 3 of thegel shown in FIG. 10. Whereas, a “cocktail” of antisenseoligonucleotides was more efficient as can be seen in section 4 of FIG.10 using a “cocktail” of antisense oligonucleotides H20A(+44+71) andH20A(+149+170). When the cocktail was used to target exon 19, skippingwas even stronger (see section 5, FIG. 10).

FIG. 11 illustrates gel electrophoresis results of exon 19/20 skippingusing “weasels” The “weasels” were effective in skipping exons 19 and 20at concentrations of 25, 50, 100, 300 and 600 nM. A further “weasel”sequence is shown in the last row of Table 3C. This compound should givegood results.

Antisense Oligonucleotides Directed at Exon 20

Antisense oligonucleotides directed at exon 20 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

None of the antisense oligonucleotides tested induced exon 20 skippingwhen delivered into cells at a concentration of 10, 25, 50, 300 or 600nM (see Table 15). Antisense molecules H20A(−11+17) [SEQ ID NO:86] andH20D(+08−20) [SEQ ID NO:87] are yet to be tested.

However, a combination or “cocktail” of H20A(+44+71) [SEQ ID NO: 81] andH20(+149+170) [SEQ ID NO:82] in a ratio of 1:1, exhibited very strongexon skipping at a concentration of 100 nM and 600 nM. Further, acombination of antisense molecules H19A(+35+65) [SEQ ID NO:79],H20A(+44+71) [SEQ ID NO:81] and H20A(+149+170) [SEQ ID NO:82] in a ratioof 2:1:1, induced very strong exon skipping at a concentration rangingfrom 10 nM to 600 nM.

TABLE 15  Antisense Ability to Oligonucloetide induce name Sequenceskipping H20A(+44+71) CUG GCA GAA UUC GAU CCA CCG GCU GUU C No skippingH20A(+149+170) CAG CAG UAG UUG UCA UCU GCU C No skipping H20A(+185+203)UGA UGG GGU GGU GGG UUG G No skipping H20A(−08+17)AUC UGC AUU AAC ACC CUC UAG AAA G No skipping H20A(+30+53)CCG GCU GUU CAG UUG UUC UGA GGC No skipping H20A(−11+17)AUC UGC AUU AAC ACC CUC UAG AAA GAA A Not tested yet H20D(+08−20)GAA GGA GAA GAG AUU CUU ACC UUA CAA A Not tested yet H20A(+44+71) &CUG GCA GAA UUC GAU CCA CCG GCU GUU C Very strong H20A(+149+170)CAG CAG UAG UUG UCA UCU GCU C skipping H19A(+44+71):GCC UGA GCU GAU CUG CUG GCA UCU UGC AGU U Very strong H20A(+44+71);CUG GCA GAA UUC GAU CCA CCG GCU GUU C skipping H20A(+149+170)CAG CAG UAG UUG UCA UCU GCU C

Antisense Oligonucleotides Directed at Exon 21

Antisense oligonucleotides directed at exon 21 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H21A(+85+108) [SEQ ID NO:92] and H21A(+85+106) [SEQ ID NO:91] inducedexon 21 skipping when delivered into cells at a concentration of 50 nM.Table 16 below includes other antisense molecules tested at aconcentration range of 5, 25, 50, 100, 200 and 300 nM. These antisensemolecules showed a variable ability to induce exon skipping

TABLE 16  Antisense Ability Oligonucleotide to induce name Sequenceskipping H21A(−06+16) GCC GGU UGA CUU CAU Skips at CCU GUG C 600 nMH21A(+85+106) CUG CAU CCA GGA ACA Skips at UGG GUC C 50 nM H21A(+85+108)GUC UGC AUC CAG GAA Skips at CAU GGG UC 50 nM H21A(+08+31)GUU GAA GAU CUG AUA Skips  GCC GGU UGA faintly to H21D(+18−07)UAC UUA CUG UCU GUA No skipping GCU CUU UCU

Antisense Oligonucleotides Directed at Exon 22

Antisense oligonucleotides directed at exon 22 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 12 illustrates differing efficiencies of two antisense moleculesdirected at exon 22 acceptor splice site. H22A(+125+106) [SEQ ID NO:96]and H22A(+80+101) [SEQ ID NO: 98] induce strong exon 22 skipping from 50nM to 600 nM concentration.

H22A(+125+146) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO:98] inducedexon 22 skipping when delivered into cells at a concentration of 50 nM.Table 17 below shows other antisense molecules tested at a concentrationrange of 50, 100, 300 and 600 nM. These antisense molecules showed avariable ability to induce exon skipping.

TABLE 17  Antisense Ability oligonucleotide to induce name Sequenceskipping H22A(+22+45) CAC UCA UGG UCU CCU No skipping GAU AGC GCAH22A(+125+146) CUG CAA UUC CCC GAG Skipping UCU CUG C to 50 nMH22A(+47+69) ACU GCU GGA CCC AUG Skipping UCC UGA UG to 300 nMH22A(+80+101) CUA AGU UGA GGU AUG Skipping GAG AGU to 50 nM H22D(+13−11)UAU UCA CAG ACC UGC No skipping AAU UCC CC

Antisense Oligonucleotides Directed at Exon 23

Antisense oligonucleotides directed at exon 23 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

Table 18 below shows antisense molecules tested at a concentration rangeof 25, 50, 100, 300 and 600 nM. These antisense molecules showed noability to induce exon skipping or are yet to be tested.

TABLE 18  Antisense Ability oligonucleotide to induce name Sequenceskipping H23A(+34+59) ACA GUG GUG CUG AGA UAG UAU AGG CC No skippingH23A(+18+39) UAG GCC ACU UUG UUG CUC UUG C No Skipping H23A(+72+90)UUC AGA GGG CGC UUU CUU C No Skipping

Antisense Oligonucleotides Directed at Exon 24

Antisense oligonucleotides directed at exon 24 were prepared usingsimilar methods as described above. Table 19 below outlines theantisense oligonucleotides directed at exon 24 that are yet to be testedfor their ability to induce exon 24 skipping.

TABLE 19  Antisense Ability oligonucleotide to induce name Sequenceskipping H24A(+48+70) GGG CAG GCC AUU CCU Needs CCU UCA GA testingH24A(−02+22) UCU UCA GGG UUU GUA Needs UGU GAU UCU testing

Antisense Oligonucleotides Directed at Exon 25

Antisense oligonucleotides directed at exon 25 were prepared usingsimilar methods as described above. Table 20 below shows the antisenseoligonucleotides directed at exon 25 that are yet to be tested for theirability to induce exon 25 skipping.

TABLE 20  Antisense Ability oligonucleotide to induce name Sequenceskipping H25A(+9+36) CUG GGC UGA AUU GUC UGA Needs AUA UCA CUG testingH25A(+131+156) CUG UUG GCA CAU GUG AUC Needs CCA CUG AG testingH25D(+16−08) GUC UAU ACC UGU UGG CAC Needs AUG UGA testing

Antisense Oligonucleotides Directed at Exon 26

Antisense oligonucleotides directed at exon 26 were prepared usingsimilar methods as described above. Table 21 below outlines theantisense oligonucleotides directed at exon 26 that are yet to be testedfor their ability to induce exon 26 skipping.

TABLE 21  Antisense Ability oligonucleotide to induce name Sequenceskipping H26A(+132+156) UGC UUU CUG UAA UUC AUC UGG AGU U Needs testingH26A(−07+19) CCU CCU UUC UGG CAU AGA CCU UCC AC Needs testingH26A(+68+92) UGU GUC AUC CAU UCG UGC AUC UCU G Faint skipping at 600 nM

Antisense Oligonucleotides Directed at Exon 27

Antisense oligonucleotides directed at exon 27 were prepared usingsimilar methods as described above. Table 22 below outlines theantisense oligonucleotides directed at exon 27 that are yet to be testedfor their ability to induce exon 27 skipping.

TABLE 22  Antisense Ability oligonucleotide to induce name Sequenceskipping H27A(+82+106) UUA AGG CCU CUU GUG CUA CAG GUG G Needs testingH27A(−4+19) GGG CCU CUU CUU UAG CUC UCU GA Faint skipping at600 and 300 nM H27D(+19−03) GAC UUC CAA AGU CUU GCA UUU C v. strongskipping at  600 and 300 nM

Antisense Oligonucleotides Directed at Exon 28

Antisense oligonucleotides directed at exon 28 were prepared usingsimilar methods as described above. Table 23 below outlines theantisense oligonucleotides directed at exon 28 that are yet to be testedfor their ability to induce exon 28 skipping.

TABLE 23  Antisense Ability oligonucleotide to induce name Sequenceskipping H28A(−05+19) GCC AAC AUG CCC AAA CUU CCU AAG v. strongskipping at 600 and 300 nM H28A(+99+124)CAG AGA UUU CCU CAG CUC CGC CAG GA Needs testing H28D(+16−05)CUU ACA UCU AGC ACC UCA GAG v. strong skipping at 600 and 300 nM

Antisense Oligonucleotides Directed at Exon 29

Antisense oligonucleotides directed at exon 29 were prepared usingsimilar methods as described above. Table 24 below outlines theantisense oligonucleotides directed at exon 29 that are yet to be testedfor their ability to induce exon 29 skipping.

TABLE 24  Antisense Ability oligonucleotide to induce name Sequenceskipping H29A(+57+81) UCC GCC AUC UGU UAG Needs testing GGU CUG UGC CH29A(+18+42) AUU UGG GUU AUC CUC v. strong UGA AUG UCG C skipping at600 and 300 nM H29D(+17−05) CAU ACC UCU UCA UGU v. strong AGU UCC Cskipping at 600 and 300 nM

Antisense Oligonucleotides Directed at Exon 30

Antisense oligonucleotides directed at exon 30 were prepared usingsimilar methods as described above. Table 25 below outlines theantisense oligonucleotides directed at exon 30 that are yet to be testedfor their ability to induce exon 30 skipping.

TABLE 25 Antisense oligonucleotide Ability to induce name Sequenceskipping H30A(+122 +147) CAU UUG AGC UGC GUC CAC CUU Needs testingGUC UG H30A(+25 +50) UCC UGG GCA GAC UGG AUG CUC Very strong skipping atUGU UC 600 and 300 nM. H30D(+19 −04) UUG CCU GGG CUU CCU GAG GCAVery strong skipping at UU 600 and 300 nM.

Antisense Oligonucleotides Directed at Exon 31

Antisense oligonucleotides directed at exon 31 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 13 illustrates differing efficiencies of two antisense moleculesdirected at exon 31 acceptor splice site and a “cocktail” of exon 31antisense oligonucleotides at varying concentrations. H31 D(+03−22) [SEQID NO:124] substantially induced exon 31 skipping when delivered intocells at a concentration of 20 nM. Table 26 below also includes otherantisense molecules tested at a concentration of 100 and 300 nM. Theseantisense molecules showed a variable ability to induce exon skipping.

TABLE 26 Antisense oligonucleotide Ability to induce name Sequenceskipping H31D(+06 −18) UUC UGA AAU AAC AUA UAC CUG UGCSkipping to 300 nM H31D(+03 −22) UAG UUU CUG AAA UAA CAU AUA CCU GSkipping to 20 nM H31A(+05 +25) GAC UUG UCA AAU CAG AUU GGA No skippingH31D(+04 −20) GUU UCU GAA AUA ACA UAU ACC UGU Skipping to 300 nM

Antisense Oligonucleotides Directed at Exon 32

Antisense oligonucleotides directed at exon 32 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H32D(+04−16) [SEQ ID NO:127] and H32A(+49+73) [SEQ ID NO:130] inducedexon 32 skipping when delivered into cells at a concentration of 300 nM.Table 27 below also shows other antisense molecules tested at aconcentration of 100 and 300 nM. These antisense molecules did not showan ability to induce exon skipping.

TABLE 27 Antisense oligonucleotide Ability to induce name Sequenceskipping H32D(+04 −16) CAC CAG AAA UAC AUA CCA CA Skipping to 300 nMH32A(+151 +170) CAA UGA UUU AGC UGU GAC UG No skipping H32A(+10 +32)CGA AAC UUC AUG GAG ACA UCU UG No skipping H32A(+49 +73)CUU GUA GAC GCU GCU CAA AAU Skipping to 300 nM UGG C

Antisense Oligonucleotides Directed at Exon 33

Antisense oligonucleotides directed at exon 33 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 14 shows differing efficiencies of two antisense molecules directedat exon 33 acceptor splice site. H33A(+64+88) [SEQ ID NO:134]substantially induced exon 33 skipping when delivered into cells at aconcentration of 10 nM. Table 28 below includes other antisensemolecules tested at a concentration of 100, 200 and 300 nM. Theseantisense molecules showed a variable ability to induce exon skipping.

TABLE 28 Antisense oligonucleotide Ability to induce name Sequenceskipping H33D(+09 −11) CAU GCA CAC ACC UUU GCU CC No skippingH33A(+53 +76) UCU GUA CAA UCU GAC GUC CAG UCU Skipping to 200 nMH33A(+30 +56) GUC UUU AUC ACC AUU UCC ACU UCA Skipping to 200 nM GACH33A(+64 +88) CCG UCU GCU UUU UCU GUA CAA UCU Skipping to 10 nM G

Antisense Oligonucleotides Directed at Exon 34

Antisense oligonucleotides directed at exon 34 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

Table 29 below includes antisense molecules tested at a concentration of100 and 300 nM. These antisense molecules showed a variable ability toinduce exon skipping.

TABLE 29 Antisense oligonucleotide Ability to induce name Sequenceskipping H34A(+83 +104) UCC AUA UCU GUA GCU GCC AGC C No skippingH34A(+143 +165) CCA GGC AAC UUC AGA AUC CAA AU No skipping H34A(−20 +10)UUU CUG UUA CCU GAA AAG AAU UAU AAU Not tested GAA H34A(+46 +70)CAU UCA UUU CCU UUC GCA UCU UAC G Skipping to 300 nM H34A(+95 +120)UGA UCU CUU UGU CAA UUC CAU AUC UG Skipping to 300 nM H34D(+10 −20)UUC AGU GAU AUA GGU UUU ACC UUU Not tested CCC CAG H34A(+72 +96)CUG UAG CUG CCA GCC AUU CUG UCA AG No skipping

Antisense Oligonucleotides Directed at Exon 35

Antisense oligonucleotides directed at exon 35 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 15 shows differing efficiencies of antisense molecules directed atexon 35 acceptor splice site. H35A(+24+43) [SEQ ID NO:144] substantiallyinduced exon 35 skipping when delivered into cells at a concentration of20 nM. Table 30 below also includes other antisense molecules tested ata concentration of 100 and 300 nM. These antisense molecules showed noability to induce exon skipping.

TABLE 30 Antisense Ability to oligonucleotide induce name Sequenceskipping H35A(+141 +161) UCU UCU GCU CGG GAG Skipping to GUG ACA 20 nMH35A(+116 +135) CCA GUU ACU AUU CAG No skipping AAG AC H35A(+24 +43)UCU UCA GGU GCA CCU No skipping UCU GU

Antisense Oligonucleotides Directed at Exon 36

Antisense oligonucleotides directed at exon 36 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

Antisense molecule H36A(+26+50) [SEQ ID NO:145] induced exon 36 skippingwhen delivered into cells at a concentration of 300 nM, as shown in FIG.16.

Antisense Oligonucleotides Directed at Exon 37

Antisense oligonucleotides directed at exon 37 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 17 shows differing efficiencies of two antisense molecules directedat exon 37 acceptor splice site. H37A(+82+105) [SEQ ID NO:148] andH37A(+134+157) [SEQ ID NO:149] substantially induced exon 37 skippingwhen delivered into cells at a concentration of 10 nM. Table 31 belowshows the antisense molecules tested.

TABLE 31 Antisense oligonucleotide Ability to induce name Sequenceskipping H37A(+26 +50) CGU GUA GAG UCC ACC UUU GGG CGU A No skippingH37A(+82 +105) UAC UAA UUU CCU GCA GUG GUC ACC Skipping to 10 nMH37A(+134 +157) UUC UGU GUG AAA UGG CUG CAA AUC Skipping to 10 nM

Antisense Oligonucleotides Directed at Exon 38

Antisense oligonucleotides directed at exon 38 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 18 illustrates antisense molecule H38A(+88+112) [SEQ ID NO:152],directed at exon 38 acceptor splice site. H38A(+88+112) substantiallyinduced exon 38 skipping when delivered into cells at a concentration of10 nM. Table 32 below shows the antisense molecules tested and theirability to induce exon skipping.

TABLE 32 Antisense oligonucleotide Ability to induce name Sequenceskipping H38A(−01 +19) CCU UCA AAG GAA UGG AGG CC No skippingH38A(+59 +83) UGC UGA AUU UCA GCC UCC AGU GGU U Skipping to 10 nMH38A(+88 +112) UGA AGU CUU CCU CUU UCA GAU UCA C Skipping to 10 nM

Antisense Oligonucleotides Directed at Exon 39

Antisense oligonucleotides directed at exon 39 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H39A(+62+85) [SEQ ID NO:153] induced exon 39 skipping when deliveredinto cells at a concentration of 100 nM. Table 33 below shows theantisense molecules tested and their ability to induce exon skipping.

TABLE 33 Antisense oligonucleotide Ability to induce name Sequenceskipping H39A(+62 +85) CUG GCU UUC UCU CAU CUG UGA UUCSkipping to 100 nM H39A(+39 +58) GUU GUA AGU UGU CUC CUC UU No skippingH39A(+102 +121) UUG UCU GUA ACA GCU GCU GU No skipping H39D(+10 −10)GCU CUA AUA CCU UGA GAG CA Skipping to 300 nM

Antisense Oligonucleotides Directed at Exon 40

Antisense oligonucleotides directed at exon 40 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 19 illustrates antisense molecule H40A(−05+17) [SEQ ID NO:157]directed at exon 40 acceptor splice site. H40A(−05+17) andH40A(+129+153) [SEQ ID NO:158] both substantially induced exon 40skipping when delivered into cells at a concentration of 5 nM.

Antisense Oligonucleotides Directed at Exon 42

Antisense oligonucleotides directed at exon 42 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 20 illustrates antisense molecule H42A(−04+23) [SEQ ID NO:159],directed at exon 42 acceptor splice site. H42A(−4+23) and H42D(+19−02)[SEQ ID NO:161] both induced exon 42 skipping when delivered into cellsat a concentration of 5 nM. Table 34 below shows the antisense moleculestested and their ability to induce exon 42 skipping.

TABLE 34 Antisense oligonucleotide Ability to induce name Sequenceskipping H42A(−4 +23) AUC GUU UCU UCA CGG ACA GUG UGC Skipping to 5 nMUGG H42A(+86 +109) GGG CUU GUG AGA CAU GAG UGA UUU Skipping to 100 nMH42D(+19 −02) A CCU UCA GAG GAC UCC UCU UGC Skipping to 5 nM

Antisense Oligonucleotides Directed at Exon 43

Antisense oligonucleotides directed at exon 43 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

H43A(+101+120) [SEQ ID NO:163] induced exon 43 skipping when deliveredinto cells at a concentration of 25 nM. Table 35 below includes theantisense molecules tested and their ability to induce exon 43 skipping.

TABLE 35 Antisense oligonucleotide Ability to induce name Sequenceskipping H43D(+10 −15) UAU GUG UUA CCU ACC CUU GUC GGU CSkipping to 100 nM H43A(+101 +120) GGA GAG AGC UUC CUG UAG CUSkipping to 25 nM H43A(+78 +100) UCA CCC UUU CCA CAG GCG UUG CASkipping to 200 nM

Antisense Oligonucleotides Directed at Exon 44

Antisense oligonucleotides directed at exon 44 were prepared usingsimilar methods as described above. Testing for the ability of theseantisense molecules to induce exon 44 skipping is still in progress. Theantisense molecules under review are shown as SEQ ID Nos: 165 to 167 inTable 1A.

Antisense Oligonucleotides Directed at Exon 45

Antisense oligonucleotides directed at exon 45 were prepared usingsimilar methods as described above. Testing for the ability of theseantisense molecules to induce exon 45 skipping is still in progress. Theantisense molecules under review are shown as SEQ ID Nos: 207 to 211 inTable 1A.

Antisense Oligonucleotides Directed at Exon 46

Antisense oligonucleotides directed at exon 46 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 21 illustrates the efficiency of one antisense molecule directed atexon 46 acceptor splice site. Antisense oligonucleotide H46A(+86+115)[SEQ ID NO:203] showed very strong ability to induce exon 46 skipping.Table 36 below includes antisense molecules tested. These antisensemolecules showed varying ability to induce exon 46 skipping.

TABLE 36 Antisense oligonucleotide Ability to induce name Sequenceskipping H46D(+16 −04) UUA CCU UGA CUU GCU CAA GC No skippingH46A(+90 +109) UCC AGG UUC AAG UGG GAU AC No skipping H46A(+86 +115)CUC UUU UCC AGG UUC AAG UGG GAU ACU AGC Good skipping to 100 nMH46A(+107 +137) CAA GCU UUU CUU UUA GUU GCU GCU CUU UUC CGood skipping to 100 nM H46A(−10 +20)UAU UCU UUU GUU CUU CUA GCC UGG AGA AAG Weak skipping H46A(+50 +77)CUG CUU CCU CCA ACC AUA AAA CAA AUU C Weak skipping

Antisense Oligonucleotides Directed at Exon 47

Antisense oligonucleotides directed at exon 47 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above. H47A(+76+100) [SEQ ID NO:170] andH47A(−09+12) [SEQ ID NO:172] both induced exon 47 skipping whendelivered into cells at a concentration of 200 nM. H47D(+25−02) [SEQ IDNO: 171] is yet to be prepared and tested.

Antisense Oligonucleotides Directed at Exon 50

Antisense oligonucleotides directed at exon 50 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

Antisense oligonucleotide molecule HSO(+02+30) [SEQ ID NO: 173] was astrong inducer of exon skipping. Further, HSOA(+07+33) [SEQ ID NO:174]and HSOD(+07−18) [SEQ ID NO:175] both induced exon 50 skipping whendelivered into cells at a concentration of 100 nM.

Antisense Oligonucleotides Directed at Exon 51

Antisense oligonucleotides directed at exon 51 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 22 illustrates differing efficiencies of two antisense moleculesdirected at exon 51 acceptor splice site. Antisense oligonucleotideH51A(+66+90) [SEQ ID NO:180] showed the stronger ability to induce exon51 skipping. Table 37 below includes antisense molecules tested at aconcentration range of 25, 50, 100, 300 and 600 nM. These antisensemolecules showed varying ability to induce exon skipping. The strongestinducers of exon skipping were antisense oligonucleotide H51A(+61+90)[SEQ ID NO: 179] and H51A(+66+95) [SEQ ID NO: 179].

TABLE 37 Antisense oligonucleotide Ability to induce name Sequenceskipping H51A(−01 +25) ACC AGA GUA ACA GUC UGA GUA GGA GC Faint skippingH51D(+16 −07) CUC AUA CCU UCU GCU UGA UGA UC Skipping at 300 nMH51A(+111 +134) UUC UGU CCA AGC CCG GUU GAA AUC Needs retestingH51A(+61 +90) ACA UCA AGG AAG AUG GCA UUU CUA GUU Very strong UGGskipping H51A(+66 +90) ACA UCA AGG AAG AUG GCA UUU CUA G skippingH51A(+66 +95) CUC CAA CAU CAA GGA AGA UGG CAU UUC Very strong UAGskipping H51D(+08 −17) AUC AUU UUU UCU CAU ACC UUC UGC U No skippingH51A/D(+08 −17) & AUC AUU UUU UCU CAU ACC UUC UGC UAG No skipping(−15+?) GAG CUA AAA H51A(+175 +195) CAC CCA CCA UCA CCC UCY GUGNo skipping H51A(+199 +220) AUC AUC UCG UUG AUA UCC UCA A No skipping

Antisense Oligonucleotides Directed at Exon 52

Antisense oligonucleotides directed at exon 52 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 22 also shows differing efficiencies of four antisense moleculesdirected at exon 52 acceptor splice site. The most effective antisenseoligonucleotide for inducing exon 52 skipping was H52A(+17+37) [SEQ IDNO:188].

Table 38 below shows antisense molecules tested at a concentration rangeof 50, 100, 300 and 600 nM. These antisense molecules showed varyingability to induce exon 50 skipping. Antisense molecules H52A(+12+41)[SEQ ID NO:187] and H52A(+17+37) [SEQ ID NO:188] showed the strongestexon 50 skipping at a concentration of 50 nM.

TABLE 38 Antisense oligonucleotide Ability to induce name Sequenceskipping H52A(−07 +14) UCC UGC AUU GUU GCC UGU AAG No skippingH52A(+12 +41) UCC AAC UGG GGA CGC CUC UGU UCC AAA Very strong UCCskipping H52A(+17 +37) ACU GGG GAC GCC UCU GUU CCA Skipping to 50 nMH52A(+93 +112) CCG UAA UGA UUG UUC UAG CC No skipping H52D(+05 −15)UGU UAA AAA ACU UAC UUC GA No skipping

Antisense Oligonucleotides Directed at Exon 53

Antisense oligonucleotides directed at exon 53 were prepared and testedfor their ability to induce exon skipping in human muscle cells usingsimilar methods as described above.

FIG. 22 also shows antisense molecule H53A(+39+69) [SEQ ID NO:193]directed at exon 53 acceptor splice site. This antisense oligonucleotidewas able to induce exon 53 skipping at 5, 100, 300 and 600 nM. A“cocktail” of three exon antisense oligonucleotides: H53D(+23+47) [SEQID NO:195], H53A(+150+175) [SEQ ID NO:196] and H53A(+14−07) [SEQ IDNO:194], were also tested, as shown in FIG. 20 and exhibited an abilityto induce exon skipping.

Table 39 below includes other antisense molecules tested at aconcentration range of 50, 100, 300 and 600 nM. These antisensemolecules showed varying ability to induce exon 53 skipping. Antisensemolecule H53A(+39+69) [SEQ ID NO:193] induced the strongest exon 53skipping.

TABLE 39 Antisense oligonucleotide Ability to induce name Sequenceskipping H53A(+45 +69) CAU UCA ACU GUU GCC UCC GGU UCU G Faint skippingat 50 nM H53A(+39 +62) CUG UUG CCU CCG GUU CUG AAG GUG Faint skippingat 50 nM H53A(+39 +69) CAU UCA ACU GUU GCC UCC GGU UCU Strong skippingGAA GGU G to 50 nM H53D(+14 −07) UAC UAA CCU UGG UUU CUG UGAVery faint skipping to 50 nM H53A(+23 +47)CUG AAG GUG UUC UUG UAC UUC AUC C Very faint skipping to 50 nMH53A(+150 +176) UGU AUA GGG ACC CUC CUU CCA UGA Very faint skipping CUCto 50 nM H53D(+20 −05) CUA ACC UUG GUU UCU GUG AUU UUC U Not made yetH53D(+09 −18) GGU AUC UUU GAU ACU AAC CUU GGU Faint at 600 nM UUCH53A(−12 +10) AUU CUU UCA ACU AGA AUA AAA G No skipping H53A(−07 +18)GAU UCU GAA UUC UUU CAA CUA GAA U No skipping H53A(+07 +26)AUC CCA CUG AUU CUG AAU UC No skipping H53A(+124 +145)UUG GCU CUG GCC UGU CCU AAG A No skipping

1-14. (canceled)
 15. An isolated antisense oligonucleotide comprisingCUUACAGGCU (base positions 14-23 of SEQ ID NO: 175), wherein theoligonucleotide specifically hybridizes to an exon 50 target region ofthe Dystrophin gene and wherein the oligonucleotide has ability to skipexon 50, wherein said uracil bases are optionally thymidine bases. 16.The isolated antisense oligonucleotide of claim 15, wherein saidoligonucleotide has ability to skip exon 50 at about 100 nMconcentration.
 17. The isolated antisense oligonucleotide of claim 15,wherein said oligonucleotide is 17-30 nucleotides in length.
 18. Theisolated antisense oligonucleotide of claim 15, wherein saidoligonucleotide induces exon skipping when tested in an assay comprisingthe steps: a. preparing myoblasts obtained from differentiated humanmuscle biopsy cultures; b. introducing the antisense oligonucleotide ofclaim 15 into the myoblasts; c. culturing the myoblasts of (b) for atleast 24 hours; d. extracting RNA; e. conducting reverse-transcriptasepolymerase chain reaction; and f. analyzing the targeted regions of thedystrophin pre-mRNA for induced exonic rearrangements.
 19. Theoligonucleotide of claim 15, further comprising an expression vector.20. The oligonucleotide of claim 19, wherein said expression vector is amodified retrovirus or a non-retroviral vector.
 21. The oligonucleotideof claim 20, wherein said non-retroviral vector is an adeno-associatedvirus (AAV).
 22. The oligonucleotide of claim 15, wherein saidoligonucleotide is 100% complementary to the target region.
 23. Theoligonucleotide of claim 15, where said oligonucleotide has beenmodified to minimize or prevent cleavage by RNaseH.
 24. A method ofinducing exon-skipping of dystrophin exon 50, comprising introducing anucleic acid molecule into a cell by way of an expression vector,wherein the nucleic acid molecule is an isolated antisenseoligonucleotide comprising CUUACAGGCU (base positions 14-23 of SEQ IDNO: 175), wherein the oligonucleotide specifically hybridizes to an exon50 target region of the Dystrophin gene and wherein the oligonucleotidehas ability to skip exon 50, wherein said uracil bases are optionallythymidine bases.
 25. The method of claim 24, wherein saidoligonucleotide has ability to skip exon 50 at about 100 nMconcentration.
 26. The method of claim 24, wherein said expressionvector is a modified retrovirus or a non-retroviral vector.
 27. Themethod of claim 26, wherein said non-retroviral vector is anadeno-associated virus (AAV).
 28. The method of claim 24, wherein thecell is a human muscle cell.
 29. The method of claim 28, wherein saidhuman muscle cell is in a patient.
 30. The method of claim 29, whereinsaid patient has muscular dystrophy.
 31. The method of claim 30, whereinsaid muscular dystrophy is Duchenne Muscular Dystrophy.
 32. The methodof claim 24, wherein said oligonucleotide is 100% complementary to thetarget region.
 33. The method of claim 24, wherein said nucleic acidmolecule and expression vector are delivered to the cell in a colloidaldispersion system.
 34. An isolated antisense oligonucleotide comprisingat least 10 nucleotides of SEQ ID NO. 175 wherein the oligonucleotidespecifically hybridizes to an exon 50 target region of the Dystrophingene and wherein the oligonucleotide has ability to skip exon
 50. 35. Amethod of inducing exon-skipping of dystrophin exon 50, comprisingintroducing a nucleic acid molecule into a cell by way of an expressionvector, wherein the nucleic acid molecule is an isolated antisenseoligonucleotide comprising at least 10 nucleotides of SEQ ID NO. 175wherein the oligonucleotide specifically hybridizes to an exon 50 targetregion of the Dystrophin gene and wherein the oligonucleotide hasability to skip exon
 50. 36. An isolated antisense oligonucleotidecomprising GAGCUCAGAUCUUCUAA (base positions 11-27 of SEQ ID NO. 174)wherein the oligonucleotide specifically hybridizes to an exon 50 targetregion of the Dystrophin gene and wherein the oligonucleotide hasability to skip exon 50, wherein said uracil bases are optionallythymidine bases.
 37. A method of inducing exon-skipping of dystrophinexon 50, comprising introducing a nucleic acid molecule into a cell byway of an expression vector, wherein the nucleic acid molecule is anisolated antisense oligonucleotide comprising GAGCUCAGAUCUUCUAA (basepositions 11-27 of. SEQ ID NO. 174) wherein the oligonucleotidespecifically hybridizes to an exon 50 target region of the Dystrophingene and wherein the oligonucleotide has ability to skip exon 50,wherein said uracil bases are optionally thymidine bases.
 38. A methodof treating Duchenne muscular dystrophy, comprising administering to apatient in need thereof an effective amount of an isolated antisenseoligonucleotide comprising CUUACAGGCU (base positions 14-23 of SEQ IDNO: 175), wherein the oligonucleotide specifically hybridizes to an exon50 target region of a Dystrophin gene and induces exon-skipping ofdystrophin exon 50, and wherein the uracil bases are optionallythymidine bases.
 39. The method of claim 38, wherein the patient is ahuman.
 40. The method of claim 38, wherein said antisenseoligonucleotide is administered as a colloidal dispersion system. 41.The method of claim 40, further comprising administering a steroid tosaid patient.
 42. A method of treating Duchenne muscular dystrophy,comprising administering to a patient in need thereof an effectiveamount of an isolated antisense oligonucleotide comprisingGAGCUCAGAUCUUCUAA (base positions 11-27 of SEQ ID NO. 174), wherein theoligonucleotide specifically hybridizes to an exon 50 target region of aDystrophin gene and induces exon-skipping of dystrophin exon 50, andwherein the uracil bases are optionally thymidine bases.
 43. A method oftreating Duchenne muscular dystrophy, comprising administering to apatient in need thereof an effective amount of an isolated antisenseoligonucleotide comprising at least 10 nucleotides of SEQ ID NO. 175,wherein the uracil bases are optionally thymidine bases.
 44. A method oftreating Duchenne muscular dystrophy, comprising administering to apatient in need thereof an effective amount of an isolated antisenseoligonucleotide comprising at least 10 nucleotides of SEQ ID NO. 175,wherein the oligonucleotide specifically hybridizes to an exon 50 targetregion of a Dystrophin gene and induces exon-skipping of dystrophin exon50, and wherein the uracil bases are optionally thymidine bases.